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An update of the Worldwide Integrated Assessment (WIA) on systemic insecticides. Part 2: Impacts on organisms and ecosystems.

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New information on the lethal and sublethal effects of neonicotinoids and fipronil on organisms is presented in this review, complementing the previous Worldwide Integrated Assessment (WIA) in 2015. The high toxicity of these systemic insecticides to invertebrates has been confirmed and expanded to include more species and compounds. Most of the recent research has focused on bees and the sublethal and ecological impacts these insecticides have on pollinators. Toxic effects on other invertebrate taxa also covered predatory and parasitoid natural enemies and aquatic arthropods. Little new information has been gathered on soil organisms. The impact on marine and coastal ecosystems is still largely uncharted. The chronic lethality of neonicotinoids to insects and crustaceans, and the strengthened evidence that these chemicals also impair the immune system and reproduction, highlights the dangers of this particular insecticidal class (neonicotinoids and fipronil), with the potential to greatly decrease populations of arthropods in both terrestrial and aquatic environments. Sublethal effects on fish, reptiles, frogs, birds, and mammals are also reported, showing a better understanding of the mechanisms of toxicity of these insecticides in vertebrates and their deleterious impacts on growth, reproduction, and neurobehaviour of most of the species tested. This review concludes with a summary of impacts on the ecosystem services and functioning, particularly on pollination, soil biota, and aquatic invertebrate communities, thus reinforcing the previous WIA conclusions (van der Sluijs et al. 2015).
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WORLDWIDE INTEGRATED ASSESSMENT OF THE IMPACT OF SYSTEMIC PESTICIDES ON BIODIVERSITYAND ECOSYSTEMS
An update of the Worldwide Integrated Assessment (WIA)
on systemic insecticides. Part 2: impacts on organisms
and ecosystems
Lennard Pisa
1
&Dave Goulson
2
&En-Cheng Yang
3
&David Gibbons
4
&
Francisco Sánchez-Bayo
5
&Edward Mitchell
6
&Alexandre Aebi
6,7
&Jeroen van der
Sluijs
8,9,10
&Chris J. K. MacQuarrie
11
&Chiara Giorio
12
&Elizabeth Yim Long
13
&
Melanie McField
14
&Maarten Bijleveld van Lexmond
15
&
Jean-Marc Bonmatin
16
Received: 25 July 2017 /Accepted: 25 September 2017
#The Author(s) 2017. This article is an open access publication
Abstract New information on the lethal and sublethal effects
of neonicotinoids and fipronil on organisms is presented in
this review, complementing the previous Worldwide
Integrated Assessment (WIA) in 2015. The high toxicity of
these systemic insecticides to invertebrates has been con-
firmed and expanded to include more species and compounds.
Most of the recent research has focused on bees and the sub-
lethal and ecological impacts these insecticides have on polli-
nators. Toxic effects on other invertebrate taxa also covered
predatory and parasitoid natural enemies and aquatic arthro-
pods. Little new information has been gathered on soil
organisms. The impact on marine and coastal ecosystems is
still largely uncharted. The chronic lethality of neonicotinoids
to insects and crustaceans, and the strengthened evidence that
these chemicals also impair the immunesystem and reproduc-
tion, highlights the dangers of this particular insecticidal class
(neonicotinoids and fipronil), with the potential to greatly de-
crease populations of arthropods in both terrestrial and aquatic
environments. Sublethal effects on fish, reptiles, frogs, birds,
and mammals are also reported, showing a better understand-
ing of the mechanisms of toxicity of these insecticides in ver-
tebrates andtheir deleterious impacts on growth, reproduction,
Responsible editor: Philippe Garrigues
*Jean-Marc Bonmatin
bonmatin@cnrs-orleans.fr
1
Utrecht University, Utrecht, The Netherlands
2
School of Life Sciences, University of Sussex, Brighton BN1 9QG,
UK
3
Department of Entomology, National Taiwan University,
Taipei, Taiwan
4
RSPB Centre for Conservation of Science, The Lodge,
Sandy, Bedfordshire SG19 2DL, UK
5
School of Life and Environmental Sciences, The University of
Sydney, 1 Central Avenue, Eveleigh, NSW 2015, Australia
6
Laboratory of Soil Biodiversity, University of Neuchâtel, Rue
Emile-Argand 11, 2000 Neuchâtel, Switzerland
7
Anthropology Institute, University of Neuchâtel, Rue Saint-Nicolas
4, 2000 Neuchâtel, Switzerland
8
Centre for the Study of the Sciences and the Humanities, University
of Bergen, Postboks 7805, 5020 Bergen, Norway
9
Department of Chemistry, University of Bergen, Postboks 7805,
5020 Bergen, Norway
10
Copernicus Institute of Sustainable Development, Environmental
Sciences, Utrecht University, Heidelberglaan 2, 3584
CS Utrecht, The Netherlands
11
Natural Resources Canada, Canadian Forest Service, 1219 Queen St.
East, Sault Ste. Marie, ON P6A 2E5, Canada
12
Aix Marseille Univ, CNRS, LCE, Marseille, France
13
Department of Entomology, The Ohio State University, 1680
Madison Ave, Wooster, OH 44691, USA
14
Smithsonian Institution, 701 Seaway Drive Fort Pierce,
Florida 34949, USA
15
Task Force on Systemic Pesticides, Pertuis-du-Sault,
2000 Neuchâtel, Switzerland
16
Centre National de la Recherche Scientifique (CNRS), Centre de
Biophysique Moléculaire, Rue Charles Sadron,
45071 Orléans, France
DOI 10.1007/s11356-017-0341-3
/ Published online: 9 November 2017
Environ Sci Pollut Res (2021) 28:11749–11797
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
and neurobehaviour of most of the species tested. This review
concludes with a summary of impacts on the ecosystem ser-
vices and functioning, particularly on pollination, soil biota,
and aquatic invertebrate communities, thus reinforcing the
previous WIA conclusions (van der Sluijs et al. 2015).
Keywords Systemicinsecticides .Neonicotinoids .Fipronil .
Insects .Pollinators .Soil biota .Aquatic organisms .
Vertebra t e s .Ecosystem services .Review
Introduction
Since the publication of the first Worldwide Integrated
Assessment (WIA) review (Bijleveld van Lexmond et al.
2015) on the impact of neonicotinoids and fipronil systemic
insecticides on invertebrates (Pisa et al. 2015), vertebrates
(Gibbons et al. 2015), ecosystem services (Chagnon et al.
2015), and its conclusions (van der Sluijs et al. 2015), there
has been a surge in publications related to this important issue.
In particular, research on the impacts of these insecticides on
bees and other pollinators has grown exponentially (Fig. 1)and
IPBES published a review report on pollinators, pollination, and
food production (IPBES 2016a), showing the great interest that
this topic has raised worldwide. In this update, we have strived to
collect all new information that has been published since 2014
onwards on the same topics covered by the WIA in 2015.
The first review paper of the updated WIA (Giorio et al.
2017, this special issue) deals with the mode of action of
neonicotinoids and fipronil, their metabolism, synergies with
other pesticides or stressors, degradation products, and the
contamination of the environment by neonicotinoids and
fipronil, including new insecticides introduced on the market.
For this second review paper, a broad-scaled literature search
was performed using the Web of Scienceand Scopus® as
reported by Gibbons et al. (2015) and restricted to the years
2014-early 2017. Search terms were [product] or
Bneonicotinoids,^and either Binsects,^Binvertebrates,^
Bvertebrates,^Bmammals,^Bbirds,^Breptiles,^Bamphibians,^
Bfish,^Bsoil biota,^Baquatic organisms,^and Becosystem
services,^where [product] was a placeholder for the name of
each considered active ingredient (a.i.): imidacloprid,
clothianidin, thiamethoxam, nitenpyram, acetamiprid,
thiacloprid, dinotefuran, cycloxaprid, imidaclothiz,
paichongding, sulfoxaflor, guadipyr, flupyradifurone, and
fipronil. In addition, specific searches were made on a few com-
mon toxicity test species (e.g., rat) and by following up refer-
ences cited in the publications found by the search. Therefore, the
present review paper covers the effects on organisms, from
aquatic and terrestrial invertebrates to vertebrates, and their im-
pacts on ecosystems.
The updated WIA is divided in three parts, corresponding
to effects on invertebrates (part A), vertebrates (part B), and
ecosystems (part C).
Note that the third paper of the updated WIA discusses the
efficacy of neonicotinoids and fipronil in agriculture and pro-
poses some alternatives to the use of these products for pest
control (Furlan et al. 2017, this special issue). It also summa-
rizes the current regulations in Europe and other countries
concerning these widely used systemic insecticides.
Part A: invertebrates
Effects of neonicotinoids and fipronil on pollinators
Honeybees (Apis mellifera)
Since the publication of the WIA document on the effects of
neonicotinoid insecticides and fipronil on non-target inverte-
brates, research on this matter has continued. Lundin et al.
(2015) provide a systematic review of research approaches,
evaluating 268 publications on bees in general (honeybees,
bumblebees, solitary bees). Another overview of scientific
advances in the field of neonicotinoids and pollinators was
made by Godfray et al. (2015). Van der Sluijs and Vaage
(2016) reviewed the implications of the present pollinator cri-
sis for global food security and concluded that it threatens
global and local food security, can worsen the problems of
hidden hunger, erodes ecosystem resilience, and can destabi-
lize ecosystems that form our life support system. They call
for an international treaty for global pollinator stewardship
that simultaneously addresses its key drivers: creation and
restoration of floral and nesting resources, a global phase out
of prophylactic use of neonicotinoids and fipronil, improve-
ment of test protocols in authorization of agrochemicals (see
Sánchez-Bayo and Tennekes, 2017 for the changes that are
needed), and restoration and maintenance of independence in
regulatory science.
0
20
40
60
80
100
120
1995 2000 2005 2010 2015 2020
Number of papers published
Fig.1 Number of research papers on pollinators and neonicotinoids
published since 1998
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In the paragraphs below, results of recent studies with re-
gard to honeybees (Apis mellifera) are listed, considering ef-
fects in vivo (field and semi-field situation) and in vitro (lab-
oratory experiments).
Field studies
Field studies to investigate effects of pesticides are observa-
tional in nature, making it hard to state causal relations be-
tween observed environmental variables and honeybee losses
or honeybee health as these are dependent on a multitude of
factors including weather, nutrition, genetics, pathogens and
diseases, presence of multiple toxic compounds, potentially
contrasting behavioral characteristics of the studied colonies,
and very different methodological approaches.
Calatayud-Vernich et al. (2016) addressed this problem by
using time series of counting dead bees in traps connected to
hives in agricultural areas(Spanishcitrusplantations),measuring
the concentration of 58 different pesticides present in dead bees
using LC-MS/MS. In this way, a change in mortality rate over
time could be correlated to a differential presence of pesticides.
The largest increases in mortality rate were associated with in-
creased presence of dimethoate and chlorpyrifos. Imidacloprid
was the fourth most present insecticide in dead bee samples, at
concentrations varying between 12 and 223 ng/g dead bees.
These concentrations are known to cause at least sublethal effects
on bees (Decourtye et al. 2005), but increased exposure to
imidacloprid presence could not be associated with bee mortality
due to the presence of other pesticides. Kasiotis et al. (2014)used
LC-ESI-MS/MS multiresidue analysis to investigate the pres-
ence and concentration of 115 pesticides in dead bees, pollen or
honey collected by bees, focusing on pollen and honey collected
by individuals or public authorities who evidenced specific high
losses or bee death incidents in 2011, 2012, and 2013. Among the
analyzed dead bees (n= 44), 50% were positive for clothianidin,
14% for chlorpyrifos, 9% for thiamethoxam, and 4.5% for
imidacloprid. Concentrations of these compounds were mostly
below the oral LD50 values for the compound detected, leading
authors to state that more research is needed to determine the
causal relations. However, the authors did not look at toxic me-
tabolites of active compounds, possibly leading to an underesti-
mation of compound presence.
An association between the presence of acetamiprid and
thiacloprid in colonies (investigated by LC-MS/MS) and suc-
cessive winter mortality was found by Van der Zee et al.
(2015). In their observational study, the presence of these pes-
ticides in any of the bee matrices (bees, pollen, wax and hon-
ey) was the second best predictor of winter loss in the ob-
served population, the first being the amount of Va r roa
destructor in colonies in October. Their results indicate that
the presenceof acetamiprid and thiacloprid in honey is a better
predictor of loss than its presence in bees and pollen. A similar
field study by Budge et al. (2015) found a correlation between
imidacloprid use in oilseed rape and colony mortality at the
landscape level. Alburaki et al. (2015) monitored hives in
neonicotinoid-treated corn areas and found elevated levels of
acetylcholine esterase gene expression (a biomarker for phys-
iological stress) in combination with higher pathogen and
Va r ro a mite loads in hives from treated locations. In a later
study, the same authors monitored colony performance and
pesticide content of foragers and trapped pollen of colonies
set up in neonicotinoid-treated corn fields and untreated corn
fields (control) (Alburaki et al. 2017). They found no
neonicotinoid compounds in foragers but sublethal amounts
of thiamethoxam and clothianidin in trapped pollen. Mogren
and Lundgren (2016), looking at pesticide presence in flowers
seeded for pollinators adjacent to crop lands, found an associ-
ation between presence of clothianidin and nutritional status
of bees. Bees with increasing amounts of clothianidin had
decreasing amounts of glycogen, lipids and protein.
Tsvetkov et al. (2017) measured long-term exposure (2 sum-
mers) to neonicotinoids in Canadian corn areas and matched their
laboratory exposure parameters to this data. They found an asso-
ciation between field-realistic exposure to clothianidin and
thiamethoxam and decreased colony immunity and survival.
Moreover, both neonicotinoids became twice as toxic in the pres-
ence of field-realistic amounts of the fungicide boscalid. Using a
large experimental design, Woodcock et al. (2017) allocated in-
secticide treatments (thiamethoxam, clothianidin, beta-
cyfluthrin, lambda-cyhalothrin), fungicide treatments (thriam,
prochloraz, fludioxonil, metalaxyl-M) and standar dized colonies
of Apis mellifera,Bombus terrestris, and units of Osmia bicornis
to a total of 33 sites with oilseed rape in the UK, Hungary, and
Germany. They found partly significant negative effects on
honeybee worker numbers and egg laying in the UK and
Hungary, but not in Germany. Their results suggest interaction
effects of treatment with the environment, available flora, and
residues of earlier treatments not part of the experiment. It
should also be noted that all treatments including controls also
received fungicide treatments and that different fungicides were
used in the three different countries. Rolke et al. (2016) carried
out a large field study of the effects of clothianidin-dressed oil-
seedrape on honey bees,findingnoadverse effect of treatment on
numbers of adult bees or brood, although the study had no repli-
cation (only one treated and one control site) and therefore these
results should be accorded little weight.
Wegener et al. (2016) have measured 28 biochemical, bio-
metrical, and behavioral aspects of honeybees (A. mellifera)to
investigate the effect of imidacloprid and fenoxycarb on col-
ony productivity and survival. Imidacloprid affected honey
yield, total number of bees, and the activity of the enzyme
phenoloxidase in worker bees.
Pilling et al. (2013) exposed hives to corn and oilseed rape
plots treated with thiamethoxam and found no effects on col-
ony parameters (mortality, colony strength, amount of brood
and honey). However, this study co-published by the
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manufacturer (Syngenta Ltd) was criticized by Hoppe et al.
(2015), who pointed to several weaknesses: the use of a non-
commercial pesticide formulation, lower than field-realistic
doses, flawed experimental design, and lack of statistical
analysis. The latter was also subject to criticism by Schick
et al. (2017) who pointed to the low quality of the data and
consequent lack of power to estimate effects.
Garbuzov et al. (2015) added to the discourse on honeybee
field studies with their findings that oil seed rape, a potential
exposure crop to neonicotinoid pesticides, elicited less forag-
ing then expected by its presence in the landscape.
An example of the requirements of large field studies and
their implementation is given by Heimbach et al. (2016). A
wider review ofneonicotinoid field studies by the industry can
be found in Schmuck and Lewis (2016). Bakker (2016)points
at shortcomings in the current field study protocols as used by
the European Food Safety Authority (EFSA) and proposes
ideas to disentangle effect measurements of acute and suble-
thal effects in experiments. Improvement of research methods
(sampling and measuring exposure) is also addressed by
Benuszak et al. (2017). Hesketh et al. (2016) provide argu-
ments for an increased exposure time (> 240 h) to better iden-
tify sublethal effects in honeybee toxicity test, the current
standard being 96 h of exposure.
A modeling approach for sublethal effects of pesticides on
colony level, using the BEEHAVE model, can be found in
Thorbek et al. (2017a). Their model study suggests that mon-
itoring of field experiments must continue for at least 1 month
to identify sublethal effects. In another publication the authors
criticize the Khoury bee population model used by EFSA to
set exposure values for colony losses related to pesticides as
tooconservative(Thorbeketal.2017b).
An interesting study that tries to bridge the gap between
field and laboratory studies has been done by Henry et al.
(2015). In their study, they show that a mixture of effects on
individual bees leads to demographic effects in the colony and
can lead to negative outcomes at the population level.
Reports on pesticide presence in dead bees generated by
investigating reported colony losses by monitoring agencies
give information about the variety and quantity of pesticides
used in the beesenvironment. A review on recent acute bee
poisonings, with a focus on Eastern Europe, is given by
Kiljanek et al. (2017). Kiljanek et al. (2016)andKimura
et al. (2014) provide information of poison incidents in a
Japanese region. A recent study in France (Daniele et al.
2017) has shown that neonicotinoids and boscalid were the
most detected pesticides in honeybees, beebread, and wax, for
numerous samples primarily taken from symptomatic colo-
nies during springs 20122016.
As stressed in the introduction to this section, observational
studies do not suffice to demonstrating causality and other
pesticides or other environmental factors may be involved in
the observed responses. However, an increasing number of
field studies include physiological and behavioral analyses
(linking field- to controlled laboratory studies) that allow more
causal interpretations of the impact of neonicotinoids on bees.
These studies converge in clearly demonstrating the existence
of a significant detrimental impact on bees.
Semi-field studies
Sandrock et al. (2014b) used a fully crossed experimental
design (sister queens, in-hive pollen feeding) to test effects
of clothianidin (2 ppb in pollen) and thiamethoxam (5 ppb in
pollen) administered during two brood cycles on colony per-
formance and queen supercedure. They found that the number
of bees and brood rearing decreased and queen supercedure
increased in treated colonies. After winter, treated colonies
exhibited a lower swarming tendency, possibly related totheir
lower growth rate. Interestingly, they found a difference in
effect for the 2 races of honeybees they used (A. m. mellifera
and A. m. carnica), with bees originating from an area with
intensive agriculture including pesticide application (A. m.
carnica)experiencing less effects of the treatments than bees
from a more natural habitat (A. m. mellifera), possibly
pointing toward a genetic adaptation. Though not a semi-
field study, the results of Rinkevich et al. (2015)alsoindicate
dramatic differences between races in sensitivity to
neonicotinoids.
In a semi-field study by Henry et al. (2015) thiamethoxam-
coated oilseed rape was sown in a specific study area (total of
288 ha in 2 years) and hives were placed at various distances
and directions to generate a range of exposure levels.
Monitoring of colony demographics showed that more ex-
posed colonies had a greater loss of forager bees, but the
numbers of foragers were buffered by colony regulation re-
sponse. However, the effects of population changes within the
beehive (larvae, nurses, workers, foragers) could weaken the
colony. Dively et al. (2015) conducted a 3-year study feeding
pollen supplements laced with imidacloprid (5, 20, and
100 μg/kg). They found an association between higher doses
(20 and 100 μg/kg) and reduced winter survival. Higher dose
colonies also had a higher Varroa mite load. Exposure to
imidacloprid and clothianidin lead to colony collapse symp-
toms at the end of winter in half of the small study population
used by Lu et al. (2014).
Tison et al. (2016) used harmonic radar to track bees at
feeders spiked with low doses of thiacloprid and unspiked
controls. They found that foraging life of bees using the spiked
feeder was shorter and that exposed bees made more naviga-
tion errors, had less homing success, and showed impaired
social communication.
Stanley et al. (2015b) tested a range of pesticides, including
acetamiprid, imidacloprid, and thiamethoxam in both labora-
tory assays (topical application and filter paper contact) and in
semi-field settings (pesticides applied to potted plants moved
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to field, application of pesticides in field directly) for toxicity
and repellent effects, using both Apis mellifera and Apis
cerana. The neonicotinoids had less direct toxicity (less lethal)
than for example deltamethrin and malathion but big differ-
ences were found between topical, filter paper, and field ap-
plications for several tested substances.
Experimental (in vitro) studies
In comparison to field and semi-field studies, where it is ex-
tremely difficult to control unwanted (and unknown) influ-
ences, properly conducted experimental assays allow for caus-
al arguments about exposure-effect relationships. Exposure of
bees to pesticides is most often done by feeding bees with
known amounts of the active substance and measuring the
lethal or sublethal responses. Whereas lethality is easily ob-
served, sublethal effects can vary greatly in their way of oc-
currence (including cascade effects) and intensity in a honey-
bee colony.
Sublethal effects on memory, behavior, and locomotion
Karahan et al. (2015) found that feeding honey bees with
field-realistic doses of imidacloprid (0.36 to 7.20 ng/bee) neg-
atively affected the number of foraging trips, number of for-
agers returning, and flowers visited. Roat et al. (2014)found
changes in the brain proteome of Africanized honeybees for
doses of 10 pg fipronil per day during 5 days. Concentrations
of several brain proteins involved in detoxification, glycolysis,
and cell growth were altered, possibly leading to memory and
learning impairment and to a reduced life span. Zaluski et al.
(2015) also used Africanized honeybees in their study about
effects of fipronil on colony development and bee motoric
control and behavior. Treated adult bees (1/500th of the
LD50) bees showed reduced motor activity and became le-
thargic, while treated colonies showed a reduction in egg lay-
ing and larval numbers.
Tan e t al. (2015) investigated the effect of imidacloprid on
adult bee memory and learning behavior by feeding total
dosesof0.24ngtolarvaeofA. cerana. They found that
long-term memory, but not short-term or larval survival, was
affected by the treatment. Also using Apis cerana in an earlier
study, these authors found that trained exposed bees foraged
less and had a lower avoidance of predators (i.e., Asian hornet
Ve s p a ve l u t i n a )(Tanetal.2014). Wright et al. (2015)useda
choice assay with imidacloprid and thiamethoxam influencing
olfactory memory. They found that low acute doses affect
olfactory memory negatively, with this effect being greater
than the effect on memory. Doses of imidacloprid (11.25 ng/
bee), clothianidin (2.5 ng/bee), and thiacloprid (1.25 mg/bee)
given to trained forager bees resulted in less successful returns
and a lower ability to navigate (Fischer etal. 2014). Effects on
learning and memory were also found by Mengoni Goñalons
and Farina (2015) who fed sublethal doses of imidacloprid to
young bees. They postulate that impaired memory and sensi-
tivity to rewards of individual bees affects colony
performance.
Peng and Yang (2016) found a reduction of mushroom
bodies in parts of the brain responsible for olfactory and visual
processing. At the molecular level, interaction between odor
binding proteins and imidacloprid has been studied in
A. cerana by Li et al. (2015a), who found that presence of
imidacloprid decreased the affinity of a specific odor binding
protein and a flower volatile.
A 24-h exposure of adult bees to imidacloprid, dinotefuran,
clothianidin, and thiamethoxam at sublethal field-realistic
doses (0.323 to 0.481 ng/bee) resulted in behavioral changes.
Bees walked less and groomed more (Williamson et al. 2014).
Blanken et al. (2015) used flight cages to determine effects of
imidacloprid (about 6 ng/mL, weekly feeding of 660 mL in a
13-week period) on flight capacity of forager bees, in combi-
nation with differential Va r ro a destructor mite loads of the bee
donor colonies. Their results showed an interaction between
physiological stressed caused by Varroa and imidacloprid,
with imidacloprid possibly affecting the body mass of bees
and lower body mass causing decreased flight capacity. An
interesting finding is that of Kessler et al. (2015). Their data
generated by choice assays (sucrose laced with imidacloprid
or thiamethoxam versus plain sucrose) suggest that bees prefer
solutions with imidacloprid and thiamethoxam. Another study
investigating effects on food consumption found that
thiamethoxam decreased beesresponse to higher sucrose
concentrations (Démares et al. 2016). Alkassab and Kirchner
(2016) exposed winter bees to sublethal doses of clothianidin
and measured behavioral effects. Chronic exposure to 15 ppb
was found to significantly affect long-term memory. Both del-
tamethrin and acetamiprid were used in retrieval assays (con-
ditional proboscis response) by Thany et al. (2015). Their
results showed that retrieval was impaired at lower doses of
acetamiprid compared to deltamethrin.
Papach et al. (2017) present the first evidence of impaired
learning and memory in adult bees that were fed
thiamethoxam (0.6 ng/bee) during the larval stage. Colony
survival critically depends on successful learning and memo-
ry. Chronic larval exposure to sublethal doses of this
neonicotinoid resulted in alterations of associative behavior
in adults. Similar delayed effects on learning and memory
following larval exposure have been reported for other
neonicotinoids such as imidacloprid (these studies are report-
ed in the WIA 2015 study).
Effects of sublethal doses of thiacloprid on social
interactions and network structure established by a group of
honeybee worker individuals has been quantified in a study by
Forfert and Moritz (2017) using experimental groups. Bees
fed with thiacloprid (0.17 and 0.80 μg thiacloprid in 20 μL
2.7 M sucrose solution) significantly reduced their network
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centrality, but they nevertheless exchanged more food to other
group members, which resulted in a dilution of the contami-
nated food. The authors argue that although thiacloprid may
act as a general perturbator of social network structure, it still
may play a role in the dynamics of disease transmission in the
colony if pathogens are transmitted via food exchange.
Using flight mills, Tosi et al. (2017) found that flight activ-
ity (duration and distance) was increased after a single suble-
thal dose and decreased (duration, distance, velocity) after 1
2 days of chronic exposure.
To understand how neonicotinoids affect behavior and
immunity at the molecular level, Christen et al. (2016)looked
at transcriptional regulation of 8 genes in caged honeybees fed
with field-realistic concentrations of acetamiprid, clothianidin,
imidacloprid, and thiamethoxam. They found downregulation
of transcription of two genes involved in memory and in-
creased transcription of the gene responsible for vitellogenin,
the latter possibly affecting foraging behavior. A follow-up
study confirmed these results and looking at effects of binary
mixtures of acetamiprid, clothianidin, imidacloprid, and
thiamethoxam on memory and vitellogenin gene transcrip-
tion, found smaller effects of mixtures opposed to single sub-
stance application on gene regulation (Christen et al. 2017).
Sublethal effects on immunity and metabolism
Gene expression profiles in honeybee midgut showed that
insecticide treatments(imidacloprid or fipronil) had no impact
on detoxifying genes but led to a significant downregulation
of immunity-related genes, suggesting a possible
immunotoxicity of neonicotinoid and phenylpyrazole insecti-
cides under chronic exposure (Aufauvre et al. 2014). This
study also showed that N. ceranae +fipronilandN. ceranae
+ imidacloprid combinations do not systematically lead to a
synergisticeffectonhoneybeemortality.Brandtetal.(2016)
found that imidacloprid, thiacloprid, and clothianidin caused
reduced hemocyte density, encapsulation response, and anti-
microbial activity after a relatively short exposure (24 h) to
field-realistic concentrations. Looking specifically at the inter-
action of thiacloprid and the pathogens Nosema ceranae and
black queen cell virus, Doublet et al. (2014) found that
thiacloprid increased the viral load of larvae and so
negatively affected larval survival, as well as aggravating the
effect of Nosema on adult mortality. A similar study by
Gregorc et al. (2016) combined exposure to Nosema ceranae
and thiamethoxam and showed no synergistic effects of the
two. Reviews of the relation between nicotinoid pesticides and
honeybee disease can be found in Sánchez-Bayo and Desneux
(2015) and Sánchez-Bayo et al. (2016b).
Badawy et al. (2015) measured the effects of oral and top-
ical application of four pesticides (acetamiprid, dinotefuran,
pymetrozine, pyridalyl) on detoxifying enzyme activity (ace-
tylcholinesterase, carboxylesterase, glutathione-S-transferase
and polyphenol oxidase). They found dinotefuran to be the
most toxic, pyridalyl second and acetamiprid/pymetrozine
the least toxic. Carboxylesterase and glutathione-S-
transferase were able to detoxify low doses of acetamiprid,
pymetrozine, and pyridalyl but not dinotefuran. Böhme et al.
(2017) were feeding pollen containing mixtures of pesticides
at field-realistic (sublethal) doses to determine synergistic ef-
fects, as exposure to multiple substances through pollen is
common but little studied. They found that larval weight
was higher and acini diameters of the hypopharyngeal
glands of nurse bees were smaller in the experimental group.
Renzi et al. (2016) also looked at hypopharyngeal glands and
found that dietary exposure to thiamethoxam was associated
with smaller acini and lower total protein content of bee heads.
Exposure to thiamethoxam was also found to alter thermo-
regulation in individual bees, with effects dependent on am-
bient temperature and dose (Tosi et al. 2016). At higher tem-
peratures (33 °C), body temperature of exposed bees in-
creased, whereas lower temperatures (22 °C) lead to lower
body temperature 6090 min post treatment. In both exposed
groups, body temperatures were lower than control group the
following day.
An interesting finding was done by Rittschof et al. (2015),
who investigated aggressive behavior of honeybees as a result
of early-life social experience, using acetamiprid as a stressor
to identify effects on the immune system. Their results found
that aggressive bees had less immunosuppressive effects of
acetamiprid than less aggressive bees.
Sublethal effects on reproduction
Sublethal effects on honeybee reproduction were not mentioned
in the original WIA article on invertebrates (Pisa et al. 2015)but
might be of considerable importance, as specific effects on, for
example, sperm viability and queen mating success might
directly affect population numbers. Williams et al. (2015) found
that queens exposed to clothianidin and thiamethoxam had larger
ovaries and reduced quality and quantity of sperm stored in the
spermatheca. Very low doses of imidacloprid, alone and in com-
bination with the parasite Nosema ceranae, were found to in-
crease activity of detoxifying enzymes and decrease survival of
queens (Dussaubat et al. 2016).
Drones that were raised in semi-field and laboratory condi-
tions and exposed to fipronil through feeding showed a decrease
in quantity of spermatozoa and increased mortality of spermato-
zoa (Kairo et al. 2017). This confirmed earlierresearchbythe
same authors had shown that queens inseminated with sperm of
fipronil exposed drones had less and less viable spermatozoa
stored in their spermatheca (Kairo et al. 2016). They found that
several pesticides, among them fipronil, imidacloprid, and
thiamethoxam, reduced sperm viability (in vitro sperm assay).
Effects on drones were also found by Straub et al. (2016), who
reported reduced drone life span as well as decreased sperm
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quality (low quantity of spermatozoa, reduced viability by 40%).
Number of newly emerged adults and drone body mass was
unaffected.Sublethaldoseof imidacloprid (2 ppb) decreased also
sperm viability by 50% 7 days after treatment in another study
(Chaimanee et al. 2016).
Wu-Smart and Spivak (2016) fed small (15007000 bees)
colonies with different doses of imidacloprid (0, 10, 20, 50,
and 200 ppb) in syrup for 3 weeks to investigate its effect on
queen productivity. They observed a decrease in egg laying
rate and queen motility associated with exposure, as well as
negative effects on foraging, hygienic behavior of worker
bees, and on colony development in all treated colonies.
Independent of colony size, number of eggs laid per 15 min
was reduced by approximately 50% by 10 ppb imidacloprid
compared to control. These findings demonstrate that chemi-
cal exposure may affect sperm quality in the spermatheca of
honey bee queens, queen fecundity, threatening the reproduc-
tive success and survival of the colony.
An interesting study on honeybee reproductive metabolism
was done by Wessler et al. (2016). They looked at the effect of
thiacloprid and clothianidin on the secretion of acetylcholine
by the hypopharyngeal gland. Acetylcholine is a key com-
pound of larval food and royal jelly. Release of acetylcholine
and its presence in larval food decreased by 80% after 4 weeks
of exposure to high doses of both neonicotinoids. Field-
realistic doses (200 ppb for thiacloprid, 1 to 10 ppb for
clothianidin) lowered acetylcholine in larval food and showed
negative effects on brood development.
Sublethal effects due to ontogenic exposure
Residue analyses of pollen, honey or bee wax revealed the
presence of a cocktail of multiple insecticides accumulating
at the same time (Bonmatin et al. 2015; David et al. 2016;
Krupke and Long 2015; Mullin et al. 2010; Daniele et al.
2017;Giorioetal.2017 this special issue). However, relative-
ly few investigations have focused on the sublethal effects of
pesticides on the honeybee brood.
It has been clearly shown that rearing brood in contaminat-
ed combs causes delayed development of larvae and emer-
gence as well as a shortened adult life span (Wu et al. 2011).
An additive interaction between black queen cell virus
(BQCV) and thiacloprid on host larval survival was also ob-
served (Doublet et al. 2014). A recent study by López et al.
(2017) demonstrated a synergistic interaction when larvae are
exposed to sublethal doses of dimethoate or clothianidin in
combination with Paenibacillus larvae, the causative agent
of American foulbrood (AFB). It is evident that the cellular
response of larvae to individual and combined stressors allows
for unmasking previously undetected sublethal effects of pes-
ticides on colony health (Giorio et al. 2017, this special issue).
Bee larvae that were fed sublethal doses of thiamethoxam
by Tavares et al. (2015a) showed condensed cells and early
cell death in the optical lobe part of the brain, as well as dose-
dependent effects on development speed and body size.
By exposing a hive to imidacloprid, Yang et al. (2012)
discovered that honey bee larvae fed with a sublethal dose
of imidacloprid still completed their development into adult
bees, but they did so with a decreased olfactory learning abil-
ity. This impairment occurred with a dose that could be as little
as 0.04 ng per larva. These results demonstrate that sublethal
dosages of imidacloprid given to the larvae affect the subse-
quent associative ability of the adult honeybee workers. Peng
and Yang (2016) further revealed the effect of sublethal doses
of imidacloprid on the neural development of the honeybee
brain by immune-labeling synaptic units in the calyces of
mushroom bodies. This not only links a decrease in olfactory
learning ability to abnormal neural connectivity but also pro-
vides evidence that imidacloprid damages the development of
the nervous system in regions responsible for both olfaction
and vision during the larval stage of the honeybee.
To reveal the potential spectrum of sublethal effects of
imidacloprid exposure in the larval stage, Wu et al. (2017)
measured changes in global gene expression in the heads of
newly emerged adults. They found that multiple physiological
changes could be induced by the sublethal exposure to
imidacloprid, affecting detoxification, immunity, sensory pro-
cessing, neuron development, metabolism, mitochondria, and
synthesis of royal jelly.
Other pollinators
Direct lethality of neonicotinoids to wild bees
Around 2000 bee species are known from Europe, with 400 of
these classified as endemic (Nieto et al. 2014). The biology,
behavior, and ecology of each of these species differ from
those of honeybees, for example, some bees ingest pollen
for transport (e.g., Hylaeus sp.), which might provide much
greater exposure than carrying pollen in corbiculae.
Consequently, extrapolating from the limited toxicological da-
ta available for 19 bee species to the effects of neonicotinoids
on the wider European fauna is fraught with difficulties given
the wide variation in relative sensitivity, ecology, and
behavioral traits. Conversely to the results of Cresswell et al.
(2012) who exposed bumble bees and honey bees to high
doses, current data suggests that wild bees are equally to
slightly less sensitive to neonicotinoids compared to honey-
bees when considering direct mortality (e.g., Sánchez-Bayo
et al. 2017). However, care must be taken when considering
individual bee species, genera, and families, as different tax-
onomic groups may show consistently different individual-
level sensitivity. Most European wild bees are smaller than
honeybees and there is the potential for them to be more sen-
sitive on a basis of a few ng/bee exposure. In general, con-
tinuing to use honeybee neonicotinoid sensitivity metrics is
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likely to be a reasonable proxy measure for the direct sensi-
tivity of the wild bee community to neonicotinoids (Arena and
Sgolastra 2014), but further work is needed in this area to
cover the wide range of bee species present in agricultural
environments.
In large parts of Asia, the ecological niche of Apis mellifera
is occupied by the similar but distinct sister species A. cerana.
As agriculture has intensified and pesticide use increased
strongly, effects can be expected on this bee species but little
toxicological research has been conducted sofar. The study of
Yasuda et al. (2017) addresses this knowledge gap. They used
the subspecies A. cerana japonica to determine LD50 values
for acute contact toxicity for commonly used pesticides. Of
the neonicotinoid group, dinotefuran proved to be most toxic
(1.4 ng/bee), followed by thiamethoxam (2.4 ng/bee),
clothianidin (3.4 ng/bee), imidacloprid (3.6 ng/bee), and
acetamiprid (278 ng/bee). This LD50 for fipronil was deter-
mined at 2.5 ng/bee. The authors note that A. cerana is gen-
erally more sensitive to pesticides and that results obtained for
A. mellifera cannot be generalized to A. cerana.
Arena and Sgolastra (2014) conducted a meta-analysis
comparing the sensitivity of wild bees to pesticides relative
to the sensitivity of honeybees. This analysis combined data
from 47 studies covering 53 pesticides from six chemical fam-
ilies with a total of 150 case studies covering 18 bee species
(plus A. mellifera). The authors calculated a sensitivity ratio
(R) between the lethal dose for species a (A. mellifera) and for
species s (other than A. mellifera), where R= LD50a/LD50s.
A ratio of over 1 indicates that the other bee species is more
sensitive to the selected pesticides than A. mellifera and vice
versa. There was high variability in relative sensitivity ranging
from 0.001 to 2085.7, but across all pesticides a median sen-
sitivity of 0.57 was calculated, suggesting that A. mellifera
was generally about two times more sensitive to pesticides
than other bee species. In the vast majority of cases (95%),
the sensitivity ratio was below 10.
Combining data for all neonicotinoids (acetamiprid,
imidacloprid, thiacloprid, and thiamethoxam) and for both
acute contact and acute oral toxicity, nine studies covering
nine bee species (plus A. mellifera) were found. These studies
showed a median sensitivity ratio of 1.045 which is the
highest median value of all the analyzed pesticide chemical
families. The most relatively toxic neonicotinoids to other
bees were the cyano-substituted neonicotinoids acetamiprid
and thiacloprid as these pesticides exhibit lower toxicity to
honeybees than the nitro-substituted neonicotinoids
imidacloprid and thiamethoxam.
In 2013, the EU installed a partial ban on imidacloprid,
clothianidin, thiamethoxam, and fipronil while allowing con-
tinued use of acetamiprid and thiacloprid. Searching for stud-
ies about effects of the banned compounds including both
acute contact and acute oral toxicity, 12 studies covering 10
bee species (plus A. mellifera) were found. These studies
showed a median sensitivity ratio of 0.957 which is close to
the calculated sensitivity ratio for all neonicotinoids. Studies
on Bombus terrestris consistently report a lower sensitivity
ratio between 0.005 and 0.914, median 0.264. Bombus
terrestris is widespread in Europe and is the most commonly
used non-Apis model system for assessing the effects of
neonicotinoids on wild bees. Differences in bee body weight
have been proposed to explain these differences, with sensi-
tivity to pesticides inversely correlated with body size
(Devillers et al. 2003). However, this has not been consistently
demonstrated and other mechanisms have been suggested
such as species-level adaptation to feeding on alkaloid-rich
nectar (Cresswell et al. 2012). With the limited data available,
Arena and Sgolastra (2014) could not comment on the
strength of these claims and further experiments are needed.
Spurgeon et al. (2016) calculated various toxicity measures of
clothianidin on honeybees, the bumblebee species B. terrestris
and the solitary bee species Osmia bicornis. Acute oral toxicity
48, 96, and 240 h LD50s for honeybees were 14.6, 15.4, and
11.7 ng/bee, respectively. For B. terrestris, the corresponding
values were 26.6, 35, and 57.4 ng/bee, respectively. For
O. bicornis, the corresponding values were 8.4, 12.4, and
28.0 ng/bee, respectively. These findings are generally in line
with the findings of Arena and Sgolastra (2014), with
B. terrestris less sensitive than A. mellifera at all time points
and O. bicornis less sensitive at 240 h.
Sgolastra et al. (2017) calculated relative sensitivity to
clothianidin to these same three species over a range of time
periodsfrom24to96h.ThehighestLD50valueswereobtained
after 24 h for A. mellifera and B. terrestris and after 72 h for
O. bicornis. At these time points, O. bicornis was the most sen-
sitive of the three species, with LD50 measurements of 1.17 ng/
bee and 9.47 ng/g, compared to 1.68 ng/bee and 19.08 ng/g for
A. mellifera and 3.12 ng/bee and 11.90 ng/g for B. terrestris.
These results are in line with the values calculated by Spurgeon
et al. (except for the 240 h values), with decreasing sensitivity in
the order of O. bicornis >A. mellifera >B. terrestris. Together,
these studies support the position that small bodied species show
greater sensitivity to neonicotinoids.
Czerwinski and Sadd (2017) found detrimental interactions
of imidacloprid exposure and bumblebee immunity. Adult
workers of Bombus impatiens received 6-day pulses of either
low (0.7 ppb) or high (7 ppb) field-realistic doses of
imidacloprid. This was followed by an assay to test immunity
and survival following a nonpathogenic immune challenge.
The results showed that high-dose imidacloprid exposure re-
duces constitutive levels of phenoloxidase, an enzyme in-
volved in melanization. Hemolymph antimicrobial activity
initially increases in all groups following an immune chal-
lenge, but while heightened activity is maintained in unex-
posed and low imidacloprid dose groups, it is not maintained
in the high exposure dose bees, although exposure had ceased
6 days prior. When imidacloprid exposure was followed by an
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immune challenge, a significantly decreased in survival prob-
ability was observed relative to control bees and those only
immune challenged or imidacloprid exposed. A temporal lag
for immune modulation and combinatorial effects on survival
suggest that resource-based trade-offs may, in part, contribute
to the detrimental interactions. These findings are particularly
relevant because such impairment of the immune system at
field-realistic exposure to neonicotinoids is likely to have
health consequences for pollinators that in real life often face
multiple stresses of sublethal neonicotinoid exposure and
pathogens. It also raises a broader question whether impair-
ment of the immune system by neonicotinoids is limited to
insects or whether it also affects other non-target species that
are exposed.
Baron et al. (2017) provides the first evidence of impacts of
thiamethoxam on the ovary development and feeding of
spring-caught wild queens of four bumblebee species:
Bombus terrestris,B. lucorum,B. pratorum,and
B. pascuorum. In a laboratory experiment testing the impacts
of field relevant doses (1.875.32 ppb) of thimethoxam, they
found that 2 weeks of exposure to the higher concentration of
thiamethoxam caused a reduction in feeding in two out of four
species, suggesting species-specific anti-feedant, repellency,
or toxicity effects. The higher level of thiamethoxam exposure
resulted in a reduction in the average length of terminal oo-
cytes in queens of all four species. Further, the authors high-
light that the discovery of species-specific effects on feeding
has significant implications for current practices and policy for
pesticide risk assessment and use.
Stingless bees (Apidae: Meliponini) are pan-tropical euso-
cial bees that are important pollinators for wild plants and
crops (Barbosa et al. 2015). Little research on exposure and
toxicology has been done for this diverse and abundant clade
that is under pressure of habitat loss and intensification of
agriculture. Lima et al. (2016) provide an overview of general
agrochemical stressors on stingless bees.
Of the available studies involving neonicotinoids or
fipronil, several indicate that the species studied are more
sensitive to certain pesticides than A. mellifera and that results
and testing procedures cannot be generalized. Topical LD50
(2.41 ng/bee 24 h, 1.29 ng/bee 48 h) and oral LC50 (2.01 ng/
μL24h,0.81ng/μL48h)valuesforMelipona scutellaris for
imidacloprid were lower than those of A. mellifera (Costa
et al. 2015). Lourenco et al. (2012) found that for fipronil
topical LD50 (0.6 ng/bee 48 h) and oral LC50 (0.011 ng/μL
48 h) were also lower than that of the honeybee. Rosa et al.
(2016) found decreased larval survival feeding field-realistic
doses (0.004 to 4.375 ng/larva) of thiamethoxam to
Scaptotrigona depilis larvae in vitro. Low doses of fipronil
(0.27 ng/bee topical, 0.24 ng/bee oral) affected brain morphol-
ogy by apoptosis or necrosis of mushroom bodies of
Scaptotrigona postica (Jacob et al. 2015), comparable to its
effect on mushroom bodies of A. mellifera (Roat et al. 2014).
Tomé et al. (2012) alsofound effects of imidacloprid on mush-
room bodies and behavior in Melipona quadrafasciata and
showed that imidacloprid impaired respiration and flight ac-
tivity in this species. Valdovinos-Núñez et al. (2009) com-
pared the toxicity of different pesticides for three stingless
bee species (Melipona beechei,Tri gona nigra,
Nannotrigona perilampoides) and found neonicotinoids
(imidacloprid, thiamethoxam, and thiacloprid) to be more tox-
ic than permethrin and diazinon.
Synergistic effects of additional pesticides with neonicotinoids
Sgolastra et al. (2017) investigated the interaction between
clothianidin and the ergosterol biosynthesis inhibiting (EBI)
fungicide propiconazole in three bee species, A. mellifera,
B. terrestris,andO. bicornis. Each species was administered
a LD10 dose of clothianidin (0.86, 1.87, and 0.66 ng/bee,
respectively, a non-lethal dose of propiconazole (7 μg/bee)
and a combination of the two treatments. Bees were then ob-
served for a 96-h period and mortality quantified. Some syn-
ergistic effects were recorded. In A. mellifera, mortality was
significantly higher for the combined dose in the first two time
periods (4 and 24 h). Mortality in B. terrestris for the com-
bined dose was only significantly higher in the first time pe-
riod, after 4 h. However, in O. bicornis, exposure to the com-
bination of clothianidin and propiconazole resulted in signif-
icantly higher mortality at all time points.
Spurgeon et al. (2016) conducted similar experiments to
Sgolastra et al., investigating the effect of a combination of
clothianidin and propiconazole on A. mellifera,B. terrestris,
and O. bicornis. In order to calculate an LD50, clothianidin con-
centrations were varied and propiconazole concentrations were
held at zero, a low dose and a high dose. The low dose was taken
from the EFSA Panel on Plant Protection Products (EFSA 2012)
reported environmental concentrations, and the high dose was 10
times the low dose to represent a plausible worst-case scenario.
Mortality was quantified over 48, 96, and 240 h. For A. mellifera,
clothianidin LD50s with and without propiconazole were always
within a factor of 2, with no clear negative trend at higher
propiconazole concentrations. For B. terrestris, clothianidin
LD50s with propiconazole were between 1.5- to 2-fold lower.
For O. bicornis, clothianidin LD50s with propiconazole was up
to 2-fold lower with a negative trend as propiconazole concen-
trations increased. Spurgeon et al. concluded that the clothianidin
and propiconazole combination had no to slight synergy for
A. mellifera and slight to moderate synergy for B. terrestris and
O. bicornis.
In an additional trial, Thompson et al. (2014)demonstrated
that the dose of fungicide applied is a key factor determining
neonicotinoid toxicity using propiconazole and thiamethoxam
mixtures. The authors argue that their low rates of significant
synergiesbetween neonicotinoidsand fungicides was because
of their lower, more field-realistic fungicide doses of 161
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447 ng/bee compared to 10,000 ng/bee used by Iwasa et al.
(2004), an early study demonstrating this interaction. The
values of 161447 ng/bee were calculated as realistic worst-
case exposures based on approved application rates for UK
crops. In a study of pesticide residues in pollen collected by
B. terrestris in the UK, David et al. (2016) found concentra-
tions of DMI fungicides up to 84 ppb, while Sanchez-Bayo
and Goka (2014) report residues of propiconazole in
honeybee-collected pollen up to 361 ppb. At the latter con-
centration a bee would need to consume approximately 28 g
of pollen to receive the dose used in the Iwasa et al. (2004)
study, which is not realistic. However, data are lacking dem-
onstrating true field-realistic exposure rates to fungicides for
free flying bees.
Overall, these studies support the position that neonicotinoids
can act synergistically with fungicides, increasing their lethality
to bees. However, the dose rate of both neonicotinoids and fun-
gicides, time of exposure, neonicotinoid and fungicide chemical
class, and length of time after exposure are all important explan-
atory factors affecting this relationship. The concentrationof fun-
gicide used in laboratory studies appears to be the most important
factor determining synergistic lethality. Fungicides are regularly
sprayed during the period when flowering crops are in bloom
under the assumption that these compounds are safe for bees.
Further work is needed in this area to establish realistic levels of
chronic exposure to fungicides for free flying bees in order to
assess the likely impact of neonicotinoid/fungicide synergies
on bee populations.
Studies to date have only examined pairwise interactions
between pesticides. It is clear that bees and other non-target
organisms inhabiting farmland are routinely exposed to far
more complex cocktails of pesticides than any experimental
protocol has yet attempted to examine (e.g., David et al. 2016;
Giorio et al. 2017 this special issue). A major challenge for
scientists and regulators is to attempt to understand how
chronic exposure to complex mixtures of neonicotinoids,
fipronil, and other chemicals affects wildlife, this withor with-
out other natural stressors (infectious agents, parasitism) and
adverse abiotic conditions.
Population-level effects of neonicotinoids on wild bees
Nothing was known about the population-level effects of
neonicotinoids on wild bees in 2014. As a managed domesti-
cated species, population trend data are available for honey-
bees, but not for wild bees. One study has attempted to inves-
tigate the impact of neonicotinoids on wild bee population
trends. Woodcock et al. (2016) used an incidence dataset of
wild bee presence in 10 × 10 km grid squares across the UK.
The dataset is comprised of bee sightings by amateur and
professional entomologists and is probably the most complete
national bee distribution database currently available. Sixty-
two wild bee species were selected and their geographic
distance and persistence over an 18-year period between
1994 and 2011 was calculated. Neonicotinoid seed-treated
oilseed rape was first used in the UK in 2002, and so the
authors calculated spatially and temporally explicit informa-
tion describing the cover of oilseed rape and the area of this
crop treated with neonicotinoids. The 62 species were split
into two groupsspecies that foraged on oilseed rape
(n= 34) and species that did not (n= 28). Species persistence
across this time period was then compared with expected
neonicotinoid exposure. Over the 18-year period, wild bee
species persistence was significantly negatively correlated
with neonicotinoid exposure for both the foraging and non-
foraging group, with the effect size three times larger for the
oilseed rape foraging group. Overall, the study suggests that
bee species were more likely to disappear from areas with a
high exposure to neonicotinoids as measured by the amounts
applied as seed dressings to oilseed rape and that this trend
was more pronounced for species known to forage on oilseed
rape. While more work is needed, this is a major correlational
study that suggests a link between levels of neonicotinoid
exposure and bee community persistence at a national scale.
Rundlöf et al. (2015) conducted an extensive field trial of
the effects of clothianidin-treated oilseed rape on wild bees.
Sixteen oilseed rape fields separated by at least 4 km were
selected across southern Sweden and were paired on the basis
of similar landscape composition. In each pair, one of the
fields was randomly selected to be sown with oilseed rape
treated with 10 g clothianidin/kg of seed and the other field
was sown without a neonicotinoid seed treatment. Twenty-
seven cocoons of the solitary bee O. bicornis (15 male, 12
female) were placed out alongside each field a week before
the oilseed rape began to flower, and six colonies of
B. terrestris were placed alongside each field on the day the
oilseed rape began to flower. The O. bicornis placed adjacent
to treated oilseed rapeshowed no nesting behavior and did not
initiate brood cell construction. O. bicornis adjacent to un-
treated fields showed nesting behavior in six of the eight fields
studied. Bumblebees placed next to treated oilseed rape
showed reduced colony growth and reproductive output.
Bumblebee colonies were collected and frozen when new
queens began to emerge, with this happening between the
7th of July and 5th of August depending on each colony.
The number of queen and worker/male cocoons present was
counted. At the point of freezing, colonies placed next to
treated oilseed rape fields had significantly fewer queen and
worker/male cocoons present.
Sterk et al. (2016) performed a similar field experiment to
Rundlöf et al. Two areas of 65 km
2
in northern Germany were
selected in which the only flowering crops comprised of
winter-sown oilseed rape. In one area, the oilseed rape was
treated with the same seed coating used by Rundlöf et al. of
10 g clothianidin/kg seed. The other area was an untreated
control. In each area, ten B. terrestris colonies were placed
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at each of six localities. Colonies were left adjacent to oilseed
rape between April and June, covering its main flowering
period. After this the colonies were moved to a nature reserve.
No differences were found in colony weight growth, number
of workers produced, or reproductive output as measured by
the production of new queens.
It is interesting to note that the latter field studies, using the
same neonicotinoid seed dressing, found markedly different
results. The major difference is that while Rundlöf et al. used
spring-sown oilseed rape, Sterk et al. used winter-sown oil-
seed rape. The length of time between sowing and peak
flowering is much greater for winter-sown oilseed rape
(mid-August to May) than for spring-sown oilseed rape
(April/May to mid-June). As such, there is more time for
neonicotinoids to degrade, and for them to leach into soil
and water for winter-sown oilseed rape, reducing the amount
of active ingredient available to be taken up by the crop.
Indeed, the mean loads of clothianidin in the Rundlöf et al.
study were 13.9 ppb in honeybee pollen, and 5.4 (bumblebee)
and 10.3 (honeybee) ppb in nectars, whereas those in the
German study were 0.500.97 ppb in honeybee pollen, 0.88
in bumblebee pollen, and 0.680.77 ppb in honeybee nectar
(Rolke et al. 2016). Such a difference as revealed by exposure
to the insecticide for honeybees (1427 times less for pollen
and 1315 times less for nectar in the latter study) could ex-
plain the difference in reported colony growth and number of
gynes and drones produced, since concentrations of
clothianidin in the food of bees below 1 ppb are not supposed
to produce any effect that were measured (Piiroinen et al.
2016). An additional difference is that in the Sterk et al.
(2016) study, colonies were moved to a nature reserve
consisting of forests, lakes, and heathland after the flowering
period of oilseed rape ended. The quality of available foraging
area at this nature reserve is likely to have been of both a
higher quality and quantity than what was available in a con-
ventional agricultural landscape and is not typical of the ex-
perience of a bumblebee colony located in such a landscape
that will have to continue foraging there after crops such as
oilseed rape cease flowering. In addition, a major problem
with the experimental design of Sterk et al. is that only one
treated and one control areawere used, so thereis no true site-
level replication, as opposed to Rundlöf et al. who used eight
treated and eight control fields. All these differences in
experimental design highlight the difficulty of developing a
single experimental design that may answer risk assessment
questions for every potentially affected species. It also
highlights the importance of evaluating the experimental
design in terms of resulting data quality when considering
the differences in results between Rundlöf et al. (2015)and
Sterk et al. (2016).
Only one study is available that looked at the impact of
neonicotinoids on the reproductive success of a solitary bee
in controlled conditions. Sandrock et al. (2014a)established
laboratory populations of O. bicornis, a solitary stem nesting
bee. Bees were fed on sugar solution treated with 2.87 ppb
thiamethoxam and 0.45 ppb clothianidin along with untreated
pollen. There was no impact of neonicotinoids on adult female
longevity or body weight. However, treated bees completed
22% fewer nests over the course of the experiment. Nests
completed by treated bees contained 43.7% fewer total cells
and relative offspring mortality was significantly higher, with
mortality rates of 15 and 8.5% in the treated and untreated
groups, respectively. Overall, chronic neonicotinoid exposure
resulted in a significant reduction in offspring emergence per
nest, with treated bees producing 47.7% fewer offspring.
These results suggest that exposure to these low-level, field-
realistic doses of neonicotinoids (< 3.5 ppb) did not increase
adult mortality but did have sublethal impacts on their ability
to successfully build nests and provision offspring.
Colony-level impact on bumblebees
Laycock et al. (2014) fed microcolonies of four B. terrestris
workers thiamethoxam-treated sugar solution at a range of con-
centrations up to 98 ppb. Pollen was not treated with
thiamethoxam. Sugar solution consumption was significantly
reduced at the 39 and 98 ppb treatments. Worker mortality was
only increased at the highest dose of 98 ppb. Worker oviposition
failure was only significantly higher at the 39 and 98 ppb treat-
ments, with no significant differences seen between the lower
concentration treatments between 0 and 16 ppb.
Scholer and Krischik (2014) exposed greenhouse
queenright colonies of Bombus impatiens to imidacloprid-
and clothianidin-treated sugar syrup at concentrations of 0,
10, 20, 50, and 100 ppb for 11 weeks. Queen mortality was
significantly increased at 6 weeks for the 50 and 100 ppb
treatments and at 11 weeks for the 20 ppb treatment for both
clothianidin and imidacloprid. Surprisingly, no significantim-
pact was found on numbers of workers or new queens pro-
duced, though this was in part because very low numbers of
new queens were produced across all treatments (average of
four per colony). Colonies in treatments above 10 ppb
imidacloprid and 20 ppb of clothianidin gained significantly
less weight over the course of the study.
Cutler and Scott-Dupree (2014)placedB. impatiens colo-
nies adjacent to maize fields during pollen shed in Ontario,
Canada. Four neonicotinoid-treated conventional and four un-
treated organic fields were used. Colonies were placed adja-
cent to each field on the first day of major pollen shed.
Colonies were left for 56 days and then transported to an
area of semi-natural habitat for 3035 days, after which they
were frozen. Colonies placed next to treated maize produced
significantly fewer workers than those placed next to organic
farms. All other metrics (colony weight, honey and pollen
pots, brood cells, worker weight, male and queen numbers
and weights) were not significantly different. However,
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bumblebees collected less than 1% of their pollen from maize
and neonicotinoid residues in collected pollen were very low
(mean of 0.4 ppb) for bees foraging adjacent to treated fields
and always below the LOD (0.1 ppb) for bees adjacent to
organic fields. Given that it is well-known that bumblebees
collect very low volumes of maize pollen, the relevance of this
study is unclear.
FERA (2013) also conducted a field trial with B. terrestris
colonies placed out adjacent to oilseed rape treated with either
clothianidin, imidacloprid or an untreated control. Colonies
were allowed to forage freely for 67 weeks while the oilseed
rape flowered and then were moved to a non-agricultural area
to continue developing. The initial aim was to measure colony
growth and development across these three treatments and
compare this with neonicotinoid concentrations collected
from food stores within the nests, but the study was criticized
for a number of methodological problems such as variable
placement date and initial colony size, lack of site-level
replication, and contamination of control colonies with
neonicotinoid residues during the experiment. The study was
ultimately not published in a peer reviewed journal but it came
to the conclusion that there was no clear relationship between
bumblebee colony success and neonicotinoid concentrations.
Goulson (2015) reanalyzed the FERA data using linear
models and retaining two colonies excluded in the original
study as outliers, but which do not meet the statistical defini-
tion of this term. This reanalysis showed that the concentration
of clothianidin in nectar (range 0 to 0.28 ppb) and the concen-
tration of thiamethoxam in pollen (range 0 to 1.6 ppb)
significantly and negatively predicted both colony weight
gain and production of new queens. Very similar findings
emerged from the recent large field trial of Woodcock et al.
(2017) who exposed B. terrestris colonies to oilseed rape
fields treated with either clothianidin, thiamethoxam or con-
trols at field sites in the UK, Germany, and Hungary. Total
neonicotinoid levels in the range 0 to 8 ppb in colony food
stores negatively predicted colony reproductive output.
Most research on neonicotinoids and bees has focused on
the three compounds subject to the EU moratorium.
Thiacloprid is considered to be less dangerous to bees, since
it has a much higher acute LD50. As a result, it is sometimes
sprayed on crops or trees at or near flowering, potentially
exposing bees to much higher doses than they would obtain
from neonicotinoids applied as seed dressings. Ellis et al.
(2017) placed B. terrestris colonies adjacent to raspberry
crops that had been sprayed with thiacloprid following normal
farming practice, and compared these to control nests placed
next to unsprayed raspberry crops. Exposed colonies were
more likely to die, grew more slowly, and produced 46%
fewer reproductive than control colonies. This study strongly
argues that thiacloprid should not be regarded as safe to bees.
Studies produced since 2014 have advanced our knowl-
edge in several key areas. Laboratory studies have continued
to demonstrate negative effects of neonicotinoids on bumble-
bee reproductive output at generally high concentrations, with
the lowest sublethal effects on reproductive output detected at
10 ppb. Field studies using bumblebees demonstrate that ex-
posure to neonicotinoid-treated flowering crops can have sig-
nificant impacts on colony growth and reproductive output
depending on the levels exposed to, with crop flowering
date relative to sowing and availability of uncontaminated
forage plants likely to explain variation in the detected
residues among the available studies. Our understanding of
the impact on solitary bees is much improved with the
findings of Sandrock et al. (2014a) suggesting substantial im-
pacts on solitary bee reproductive output at field-realistic con-
centrations of 3.5 ppb. Field studies demonstrating this under
real-world conditions are limited with the work of Rundlöf
et al. (2015) and Woodcock et al. (2017), demonstrating no
nest-building activity at the neonicotinoid treatment sites.
Feltham et al. (2014)exposedB. terrestris colonies to sugar
solution treated with 0.7 ppb and pollen treated with 6 ppb of
imidacloprid for 2 weeks. Colonies were then placed out in an
urban area in Scotland. The foraging workers from each nest
were then monitored for a further 4 weeks. There was no
difference in the length of time spent collecting nectar or the
volume of nectar collected between workers from treated and
control colonies. However, treated workers collected signifi-
cantly less pollen, bringing back 31% less pollen per time unit
to their colonies. Treated workers also collected pollen less
frequently, with 41% of foraging bouts collecting pollen ver-
sus 65% for control workers; a decline of 24%.
Gill and Raine (2014) performed an experiment where
B. terrestris colonies were exposed to sugar solution treated
with 10 ppb of imidacloprid while also having access to forage
freely outside. Colonies and individual worker bumblebees
were studied over a 4-week period. In common with their
previous findings (Gill et al. 2012), imidacloprid-treated
workers initiated significantly more foraging trips across all
4 weeks of the experiment. The authors note that this is likely
driven by an acute individual-level response in the first weeks
(neonicotinoids acting as a neural partial agonist, increasing
desire to forage) and by a chronic colony-level response in the
latter part ofthe experiment, with treated colonies allocating a
higher proportion of workers to pollen collection. Pollen for-
aging efficiency of treated workers decreased as the experi-
ment progressed with the smallest collected pollen loads re-
corded in week four, suggesting a chronic effect of
imidacloprid on pollen foraging ability. It is not clear whether
this is as a result of individual performance deteriorating or
new emerging workers having been exposed for a greater
period of time.
Stanley et al. (2015a) exposed B. terrestris colonies to 2.4 or
10 ppb thiamethoxam-treated sugar solution for 13 days.
Colonies were then moved to pollinator exclusion cages where
they were allowed to forage freely on two varieties of apple
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blossom. Bees from colonies exposed to 10 ppb spent longer
foraging, visited fewer flowers and brought back pollen on a
lower proportion of foraging trips compared to bees from
control colonies. Stanley and Raine (2016) also exposed
B. terrestris colonies to 10 ppb thiamethoxam sugar solution
for a 9- to 10-day period. At this point, colonies were moved to
a flight arena provisioned with two common birds-foot trefoil
(Lotus corniculatus) and one white clover (Trifolium repens)
plants. Worker bees were individually released and their interac-
tion with the flowers was recorded. Significantly more treated
workers displayed pollen foraging behavior compared to control
workers. However, control workers learned to handle flowers
efficiently after fewer learning visits.
Arce et al. (2016)placedB. terrestris nests out in an area of
parkland for a 5-week period while also supplying them with
sugar solution treated with 5 ppb of clothianidin. The volume
of sugar solution provided was estimated to be half that which
colonies typically consume over the course of the experiment.
No pollen was provided, so workers had to forage for this and
to make up the shortfall in nectar resources. In contrast to the
previous papers, only subtle changes to patterns of foraging
activity and pollen collection were detected. There was no
clear difference in colony weight gain between treatments or
number of brood individuals. However, by the end of the
experiment, treated colonies contained fewer workers, drones,
and gynes when compared with control colonies.
Switzer and Combes (2016) studied the impact of acute
imidacloprid ingestion on sonicating behavior of
B. impatiens. Sonicating is a behavior whereby a bumblebee
lands on a flower and vibrates loudly to shake pollen loose
from anthers. Bumblebee workers were fed a dose of 0,
0.0515, 0.515, or 5.15 ng of imidacloprid in 10 μLofsugar
solution. These are equivalent to concentrations of 0, 5.15,
51.5, and 515 μg/L (~ppb), with the highest volume con-
sumed equivalent to 139% of the honeybee acute LD50, a
moderate proxy for bumblebees, as bumblebees are generally
less sensitive than honeybees. Bees were then allowed to for-
age from tomato (Solanum lysopersicum) plants and sonicat-
ing behavior was observed. At the lowest dose of 5.15 μg/L of
imidacloprid, no impact was found on wingbeat frequency,
sonication frequency or sonication length. No analysis could
be made for higher doses, as bees in these treatments rarely
resumedforagingbehaviorafter ingesting imidacloprid.
Given the neonicotinoid concentrations used in this study
and sample size problems, it is difficult to draw many conclu-
sions other than that levels of exposure above 50 μg/L impair
bumblebee pollen foraging behavior.
Overall, these studies suggest that exposure to neonicotinoids
in nectar at concentrations between 0.710 ppb can have suble-
thal effects on the ability of bumblebees to collect pollen at both
the individual and colony level. This shortfall in pollen and sub-
sequent resource stress is a plausible mechanism to explain di-
minishedcolonygrowth and production of sexuals in the absence
of increased direct worker mortality.G iven that concentrations as
high as 10 ppb are at, but within, the upper limit of what bumble-
bees are likely to experience in the field, it is likely that wild
bumblebees exposed to neonicotinoids in contemporary agricul-
tural environments suffer from a reduced ability to collect pollen,
with a subsequent impact on their reproductive output.
Effects of neonicotinoids and fipronil on other
invertebrates
Effects on target pests
Fipronil induced Drosophila S2 cell apoptosis in vitro exper-
iments (Zhang et al. 2015). This side effect occurs through
caspase-dependent mitochondrial pathways and appears to
coincide with a decrease in the mitochondrial membrane po-
tential and an increase in reactive oxygen species. Other au-
thors have shown significant increases in tumor frequencies
on wing cells of Drosophila melanogaster, suggesting that
this insecticide is mutagenic and carcinogenic in somatic cells
of this fruit fly (de Morais et al. 2016a).
Wild strains of Drosophila melanogaster are rather resis-
tant to imidacloprid with acute LD50s > 1304 μM
(> 333.8 ppm) for both females and males (Charpentier et al.
2014). However, the same study has shown lethal effects of
imidacloprid on chronically exposed D. melanogaster during
8 days: 27% of females died at 3.91 nM and 28% of males at
39.1 nM.The latter concentrations were several orders of
magnitude below chronic LC50s of 18and 45 μM for females
and males, respectively. Moreover significant sublethal effects
have been demonstrated on mating and fecundity at very low
exposure concentrations (mating: both genders exposed at
0.391 nM; fecundity: females exposed at 3.91 nM), i.e., in
the 0.11 ppb range of concentration.
Stimulated reproduction of the green peach aphid (Myzus
persicae) by exposure to sublethal doses of imidacloprid had
been reported previously (Yu et al. 2010). This hermetic effect
undermines the effectiveness of the insecticide in controlling
the target pest, and it seems to be accompanied by a complex
pattern of up- and downregulation of genes during exposure.
A recent study suggests that such an effect is passed on to the
second generation, although there is some adaptability to low
doses of the insecticide (Ayyanath et al. 2014). In another
study, the soybean aphid (Aphis glycines) showed significant-
ly higher reproduction rate when treated at sublethal doses of
imidacloprid (0.05 mg/L) than in non-treated controls (Qu
et al. 2015). However, other sublethal doses (0.1 and
0.2 mg/L) caused slower juvenile development, shorter repro-
ductive periods, and reduced adult lifespan and fecundity, in-
dicating that the threshold for hormetic responses is rather
low. Stimulatory reproductive effects have also been observed
with exposure of males of the Neotropical brown stink
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(Euschistus heros) to imidacloprid, but not with exposure of
the females (Haddi et al. 2016).
A commercial mixture of a pyrethroid (b-cyfluthrin) and a
neonicotinoid (imidacloprid) produced behavioral sublethal
effects on bed bugs (Cimex lectularius) such as reduced loco-
motion, reduced feeding, and difficulties in host-finding that
resulted in good control of the bugs by preventing their dis-
persal (Crawley et al. 2016). However, the bugs have already
developed resistance to at least four neonicotinoids,
acetamiprid, dinotefuran, imidacloprid, and thiamethoxam in
several North American cities (Romero and Anderson 2016).
Efficacy of three neonicotinoids (acetamiprid,
imidacloprid, and thiamethoxam) oncontrolling sand termites
(Psammotermes hypostoma; Isoptera) has been demonstrated
by Ahmed et al. (2015), the effect of all three lasting up to
60 days. Dembilio et al. (2015) also studied the lasting effect
of imidacloprid applied by crown spray or stipe injection on
palms to control the red palm weevil (Rhynchophorus
ferrugineus; Coleoptera). Complete control (100%) was
achieved after 45 days of stipe injections at 410 mL, corre-
sponding to 2 g a.i./plant. Spray application used larger vol-
umes that stipe injections, were less efficient and resulted in
larger losses of the insecticide by washoff from the palm
fronds into the surrounding environment. In Brazil, planta-
tions of eucalypts were treated by immersing the seedlings
in a solution containing fipronil (0.4%) to prevent attack by
termites, as it has been shown to be protective for 56 days (dos
Santos et al. 2016). A comparison of termitesdiversity be-
tween treated and untreated plots did not show significant
differences, although treated plots tended to have fewer spe-
cies (Silva et al. 2016). The authors indicated that Bany effect
[of fipronil] is masked by the effects of the plantation itself^,
since both treated and untreated plantations of this tree had
significantly less termite diversity than native savanna forests
or regeneration forests.
Baits laced with fipronil are being used to control the ex-
pansion of the invasive Argentine ant (Linepithema humile)in
Japan. Although the main super colonies of this species appear
to be very susceptible to this insecticide, the treatment was
also damaging for all other local ant populations. Fipronil bait
treatments, therefore could lead to significant impacts on the
local arthropod biodiversity (Hayasaka et al. 2015). Hydrogels
containing 750 ppb thiamethoxam killed 50% of the forager
ants in 3 days, while baiting with 1500 and 750 ppb provided
100% mortality of workers and queens within 8 days in the
laboratory trials. These concentrations were lower than the
ones required to control ants in highly infected areas as report-
ed by Rust et al. (2015). At sublethal levels, imidacloprid may
have different effects in red imported fire ants (Solenopis
invicta) depending on the concentrations used; for example,
concentrations of this insecticide in sugar water at 0.01 ng/L
are attractive to the ants and increase their digging activity,
whereas concentrations higher than 0.25 ng/L suppress their
water consumption, digging and foraging behaviors (Wang
et al. 2015e). At the latter concentration, newly mated queens
reduced their brood tending ability, while the time to larval
emergence was delayed significantly and no pupae or adult
workers were produced (Wang et al. 2015d).
Effects on butterflies
Mulé et al. (2017) presented a systematic review of the effects
of chemical insecticides on four common butterfly families:
Lycaenidae,Nymphalidae,Hesperiidae, and Papilionidae.
Only one study in their sample (Krischik et al., 2015)looked
at the effects of a neonicotinoid (imidacloprid) on butterflies
(Danaus plexippus and Vanessa cardui) illustrating a huge
data gap. The systematic review concludes that the use of all
the insecticides studied (dichlorvos, imidacloprid, malathion,
naled, permethrin, and resmethrin) cause negative effects on
the most common butterfly families, such as reduced survival
rate, feeding interruption, and alteration of oviposition
behavior.
Effects on natural enemies of pests
Compared to the research on pollinators, few studies on the
toxic effects and population impacts of neonicotinoids and
fipronil on other arthropods have been published in the past
2 years. Research in this case has been limited to beneficial
insects used in biological control or integrated management
programs (IPM), building upon the already known negative
effects of these chemicals (Pisa et al. 2015). Recent studies
have centered on the newly developed compounds (Giorio
et al. 2017, this special issue), while the previous literature
focused mainly on imidacloprid.
Predators
The efficacy of neonicotinoids and fipronil for pest control
and the negative effects they inflict on beneficial predators
are directly correlated. Both effects depend on the toxicity to
pest and to theirpredators and on the residue level of exposure
in plants (which have been treated or not). It has been shown
that uptake of several neonicotinoids after seed treatment in
cotton crops differed according to their water solubility, with
nitenpyram, dinotefuran, and thiamethoxam showing the
highest residues in plant tissues and lowest in the soil
(Zhang et al. 2016b). Consequently, these three compounds
were more effective against the cotton aphid Aphis gossypii
than the other four neonicotinoids. However, residues in soil
of all seven neonicotinoids reduced the soil fauna significantly
(p< 0.05), in particular the larvae of hoverflies (Diptera:
Syrphidae). Foliar sprays of all compounds produced similar
effects to their seed treatments but the impacts on soil larvae
werenotsignificant(Zhangetal.2016b). The authors
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acknowledge the efficient aphid control by the three
neonicotinoids above while warning of the long-term negative
effects derived of suppressing beneficial insectslarvaeand
also species that feed on extra floral nectar, such as some
ladybugs and polyphagous parasitoids. Another study sug-
gests that not only are there no significant negative impacts
of seed-treated imidacloprid and clothianidin onthe beneficial
insects of winter wheat crops (i.e., ladybirds, hoverflies, or
parasitoids) but both systemic treatments increased the density
of spiders, despite residues in plants being present for 200 days
(Zhang et al. 2016a). The latter field study was conducted
throughout the winter season in northern China, with planting
in October and harvest in June, when the soil larvae were
dormant or going through diapausehence the lack of nega-
tive effects, contrasting with those observed in summer crops
and the high toxicity of neonicotinoids to coccinelid larvae
(Lucas et al. 2004).
The variability of impacts on natural enemies of crop pests
depends on the intrinsic toxicity of the active ingredients and
co-formulants, and the rate of application to the crop. Thus,
cotton plants grown from seeds treated with thiamethoxam at
the recommended doses (3 g/kg) reduced the populations of
natural enemies of the cotton leafhopper by about 35%, in
particular those of Chrysoperla sp., Orius sp., and spiders,
whereas cotton plants grown from seeds treated with
imidacloprid (5 g/kg) did not lead to significant reductions
(i.e., < 10%) of the same species (Saeed et al. 2016). Trials
in South Dakota demonstrated the effectiveness of seed treat-
ments of thiamethoxam and imidacloprid, whether alone or in
combination with foliar sprays of beta-cyflutrin, for control-
ling aphids. However, thrips (Thysanoptera) increased in
number in one of the locations regardless of the treatment
used, and this effect was significantly correlated with their
major predators, as several taxa of natural enemies declined
(Regan et al. 2017).
Polyphagous ladybird beetles like Coleomegilla maculata
and Hippodamia convergens feed occasionally on the nectar
of sunflowers. Three routes of exposure of thiamethoxam for
the ladybird Serangium japonicum that controls the whitefly
Bemisia tabaci were tested. Predation of S. japonicum was
most reduced under systemic exposure and least by contact
with residues on the surface, following the same pattern as the
lethal toxicity (Yao et al. 2015). In a similar way, oral exposure
of the ladybird Eriopis connexa to acetamiprid at maximum
recommended rates (200 mg/L) in water resulted in 90% mor-
tality of adults after 15 days, whilethe survival of pupae treat-
ed in Petri dishes at half that rate (100 mg/L) was reduced only
up to 15%. However, 83% of the emerging adults had a num-
ber of malformations (Fogel et al. 2016). Residues of
imidacloprid and thiamethoxam on filter paper appear to repel
the predatory beetles Cycloneda sanguinea and
Chauliognathus flavipes as well as the predatory bug Orius
insidiosus (Fernandes et al. 2016), but tomato leaves and
plants treated with foliar sprays of imidacloprid at 100 ppm
had residual activity and caused 62% mortality of the mirid
bug Macrolophus basicornis a month after treatment
(Wanumen et al. 2016a).ThelifespanofCoccinella
septempunctata adults exposed to sublethal doses of
imidacloprid sprayed on leaves (4.8 ppm) was reduced by
2428%, while their fecundity was reduced by 5356% and
the oviposition period was shortened significantly. Moreover,
the fecundity of the F1 generation was also reduced consider-
ably (Xiao et al. 2016). Foliar sprays of imidacloprid on okra
crops in India at recommend label rates (2124.5 g/ha) re-
duced the populations of spiders and ladybeetles significantly
in the first 2 weeks after spraying, but the authors concluded
that the insecticide was safe to natural enemies because they
recovered after a while (Karthik et al. 2015).
Unfortunately, the rates of application of neonicotinoids in
greenhouses, nurseries, and trees in urban landscapes are
much higher than the rates applied to field crops. Thus,
imidacloprid applied at 300 mg/L to pots containing the
Mexican milkweed (Asclepias curassavica)resultedinvery
high concentrations of 6 ppm in the flowers after one applica-
tion and 21 ppm after a second one done 7 months later.
Consequently, the concentrations of imidacloprid in pollen
of the nursery flowers were 793 to 1368 times higher than
the typical residues (7.6 ppb) obtained from seed-treated ca-
nola plants. Such residue levels caused significant mortality in
three lady beetle species after 12 days, Coleomegilla maculata
(5065%), Harmonia oxyridis (2550%), and Hippodamia
convergens (3050%), but less in Coccinella septempunctata
(~ 1015%). Caterpillars of Danaus plexippus fed on the same
plants experienced > 90% mortality after 1 week and were
wiped out after 3 weeks. Vanessa cardui butterflies fed on
flowers of globe thistle (Echinops ritro) treated at the same
rate experienced mortalities of over 30% after 1 week com-
pared to untreated controls (Krischik et al. 2015).
In field experiments, sunflowers grown from seeds treated
with thiamethoxam at the recommended dose (0.5 mg/kg
seeds) did not cause significant mortality of the predatory
bug Orius insidiosus, but reduced its egg viability and female
fertility resulting in a 40% reduction in nymph survival
(Gontijo et al. 2015). The same treatment, however, caused
48% mortality of the predator Chrysoperla carnea after 8 days
exposure (Gontijo et al. 2014). Thiamethoxam applied as seed
treatment also delayed emergence of Coleomegilla maculata
by prolonging the pupal period, whereas it reduced egg via-
bility and skewed the sex ratio of Hippodamia convergens in
favor of females (Moscardini et al. 2015).
Secondary poisoning has been shown with second instars
of the ladybug Coleomegilla maculata, which have slower
walking and predatory skills when feeding on aphids
(Rhophalosiphum padi) grown on wheat plants seed-treated
with thiamethoxam. Interestingly, only residues of its metab-
olite clothianidin were found in the aphids (Bredeson et al.
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2015). The omnivorous mirid predator Nesidiocoris tenuis
experienced a mortality of 36% when feeding on eggs of
Ephestia kuehniella (Lepidoptera, Pyralidae) that had been
treated with sulfoxaflor at the highest recommended rates
(60 mg/L) for controlling this pest. In addition, fecundity
and longevity of the predatory bug were reduced significantly,
indicating that this member of the fourth generation of
neonicotinoids has undesirable sublethal effects on natural
enemies (Wanumen et al. 2016b).
An update on the acute toxicity of seven neonicotinoids
and fipronil to predatory arthropods is shown in Table 1.As
most of the authors cited above have indicated, the sublethal
effects of thiamethoxam on reproduction and predation ability
of natural enemies and the residual activity of imidacloprid do
not warrant the use of these insecticides in IPM programs.
Parasitoids
ABmeta-analysis of nearly 1,000 observations from North
American and European field studies revealed that seed-
applied neonicotinoids reduced the abundance of arthropod
natural enemies similarly to broadcast applications of pyre-
throid insecticides^(Douglas and Tooker 2016). The study
also indicates that seed-applied neonicotinoids are less toxic
than pyrethroids to spiders and mites, so they might contribute
to biological control in some particular agricultural systems.
Imidacloprid, dinotefuran, and thiamethoxam were more tox-
ic to the filth fly parasitoid Spalangia endius (Hymenoptera:
Pteromalidae) than to the target pest (Musca domestica), thus
making it unsuitable for controlling the flies (Burgess and King
2015). Also S. endius was attracted to imidacloprid granular baits
and experienced more grooming activity, but not to those con-
taining dinotefuran or other insecticides. By contrast, another
Pteromalidae parasitoid, Urolepis rufipes, did not show that be-
havior with either neonicotinoid tested (Burgess and King 2016).
In semi-field tests designed to evaluate the toxicity of 19 new
insecticides applied at label ratestocottonontheeggparasitoid
Trichogramma pretiosum, fipronil (480 ppm) and dinotefuran
(1040 ppm) showed almost 100% mortality in 24 h, whereas
acetamiprid (429 ppm) caused 80% mortality (Khan et al.
2015). Similarly, imidacloprid applied at the label rate on citrus
trees (40 ppm) is harmful to the encyrtid parasitoid Ageniaspis
citricola, causing over 89% mortality in 24 h and having residual
activity on the leaves up to 17 days (de Morais et al. 2016b).
Adults of the parasitoid Tamarixia triozae (Eulophidae) experi-
enced 28 to 58% mortality after contact with pepper leaves that
had been sprayed with imidacloprid (range 3 to 260 ppm); the
highest dosage also caused cumulative 100% mortality after
2 days, whereas the lower treatments reduced emergence by
2663% in a dose-related manner (Martinez et al. 2015).
However, residues of imidacloprid on sprayed tomato leaves
(1155 ppm) caused only 38% mortality in 24 h to the parasitoid
Tamarixia triozae (Eulophidae), although its residual activity
after 11 days was still noticeable and still caused 25% mortality
(Luna-Cruz et al. 2015).
Laboratory tests using glass residues (contact toxicity) have
demonstrated the high toxicity of imidacloprid, dinotefuran,
nitenpyram, and thiamethoxam to the egg parasitoid
Trichogramma ostrinae (Table 2), and all neonicotinoids ex-
cept nitenpyram appear to pose a high risk in IPM: the fecun-
dity is reduced by more than 50% in the case of thiamethoxam
and dinotefuran, while emergence is reduced 54% in the case
of imidacloprid exposure (Li et al. 2015b). In the case of
Trichogramma chilonis exposed to residues of commercial
formulations of thiamethoxam and nitenpyram, mortalities
of 98 and 96%, respectively, were observed in 24 h. This
together with a reduction of parasitism in the larval stage:
2037% for thiamethoxam and 1445% for nitenpyram, and
a reduced emergence of 1233% for thiamethoxam and 21
29% for nitenpyram (Ko et al. 2015). Also, exposures by
contact following the recommended paper disc tests of the
International Organization for Biological Control (IOBC)
showed the high acute toxicity of thiamethoxam, imidacloprid
and acetamiprid when applied at 25, 3040, and 60 ppm con-
centrations. Adult mortalities of the parasitoid Tam a rix i a
radiata after 3 days were 100, 6178, and 66% for the respec-
tive insecticides (Beloti et al. 2015). Therefore, these insecti-
cides were classified in Class 4 (harmful) and not recommend-
ed for IPM programs (Veire et al. 2002).
Furthermore, females of the chalcid wasp Nasonia
vitripennis exposed to sublethal concentrations of
imidacloprid in syrup (2100 ppb) not only experienced 20
25% reduced fecundity but also altered the sex allocation of
the offspring in favor of females while reducing the fitness
when ovipositing with co-foundresses (Whitehorn et al.
2015). The reproductive impairment observed so far with var-
ious species of parasitoid wasps is evidence that links
neonicotinoids to the decline of these beneficial and important
species for pest control.
Effects on non-target soil organisms
Imidacloprid and thiacloprid are highly toxic to springtails
(Collembola spp.), with LC50s of 0.44 and 9 mg/kg dry soil,
respectively, for Folsomia candida. In multigenerational tests
with this species, imidacloprid showed consistently high tox-
icity through three generations, whereas toxicity of thiacloprid
was reduced in the second and third generation. The authors
suggest that the higher persistence of imidacloprid in soil com-
pared to that of thiacloprid could be the reason for this differ-
ential toxicity in time (van Gestel et al. 2017).
The only microcosm study available for terrestrial arthro-
pods using imidacloprid was done by Uhl et al. (2015). The
experimental setup consisted of a tritrophic system: strawber-
ry plants, a ground cricket (Nemobius sylvestris)andaweb
spider (Pisaura mirabilis). Strawberry leaves were treated at
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Tab l e 1 Lethal median concentrations (LC
50
, mg/L) of systemic insecticides to predatory arthropods of crop pests
Scientific name Taxon Acetamiprid Clothianidin Dinotefuran Imidacloprid Thiacloprid Thiamethoxam Fipronil References
Neoseiulus fallacis Acari: Phytoseiidae 57 Lefebvre et al. (2012)
Phytoseiulus macropilis Acari: Phytoseiidae 3561 Mizell and Sconyers (1992)
Gnathonarium exsiccatum Arachnida: Linyphiidae 801 Tanaka et al. (2000)
Ummeliata insecticeps Arachnida: Linyphiidae 995 Tanaka et al. (2000)
Pardosa pseudoannulata Arachnida: Lycosidae 440 Tanaka et al. (2000)
Pardosa pseudoannulata Arachnida: Lycosidae 40.4 Chen et al. (2012)
Tetragnatha maxillosa Arachnida: Tetragnathidae 136 Tanaka et al. (2000)
Chauliognatus flavipes Coleoptera: Cantharidae 80* 470* (Fernandes et al. 2016)
Adalia bipunctata Coleoptera: Coccinellidae 218.9 232 Amirzade et al. (2014)
Adalia bipunctata Coleoptera: Coccinellidae 74 Jalali et al. (2009)
Cheilomenes quadriplagiata Coleoptera: Coccinellidae 307 Wu et al. (2007)
Coccinella septempunctata Coleoptera: Coccinellidae 35.8 Xue and Li (2002)
Coccinella septempunctata Coleoptera: Coccinellidae 726 Bozsik (2006)
Coccinella undecimpunctata ssp. aegyptica Coleoptera: Coccinellidae 93.5 34.2 Ahmad et al. (2011)
Coccinella undecimpunctata ssp. aegyptica Coleoptera: Coccinellidae 263.4 447.8 296.6 Amirzade et al. (2014)
Cryptolaemus montrouzieri Coleoptera: Coccinellidae 20.6 Khani et al. (2012)
Cycloneda sanguinea Coleoptera: Coccinellidae 760* 420* Fernandes et al. (2016)
Harmonia axyridis Coleoptera: Coccinellidae < 416.7 30.3364 153.3 Youn et al. (2003)
Hippodamia convergens Coleoptera: Coccinellidae 161.4 164.3 Kaakeh et al. (1996)
Hippodamia variegata Coleoptera: Coccinellidae 788.5 Rahmani and Bandani (2013)
Olla v-nigrum Coleoptera: Coccinellidae 3.07 Mizell and Sconyers (1992)
Propylaea japonica Coleoptera: Coccinellidae 629 Wu et al. (2007)
Propylaea sp. Coleoptera: Coccinellidae 12.4 Xue and Li (2002)
Serangium japonicum Coleoptera: Coccinellidae 2.43 Yao et al. (2015)
Stethorus japonicus Coleoptera: Coccinellidae 0.6 Mori and Gotoh (2001)
Orius insidiosus Hemiptera: Anthocoridae 2.78 1.67 Prabhaker et al. (2011)
Orius insidiosus Hemiptera: Anthocoridae 80* 380* Fernandes et al. (2016)
Orius laevigatus Hemiptera: Anthocoridae 0.040.3 Delbeke et al. (1997)
Geocoris punctipes Hemiptera: Lygaedae 5180 2170 Prabhaker et al. (2011)
Cyrtorhinus lividipennis Hemiptera: Miridae 0.36 Tanaka et al. (2000)
Cyrtorhinus lividipennis Hemiptera: Miridae 0.043 0.94 Preetha et al. (2010)
Deraeocoris nebulosus Hemiptera: Miridae 0.0163 Mizell and Sconyers (1992)
Hyaliodes vitripennis Hemiptera: Miridae 0.7 1.1 0.3 0.5 Bostanian et al. (2005);
Bostanian et al. (2001)
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two different rates that reflected a typical dosage within the
crop (2.4 g/m
2
) and low exposure in field margins and forests
(0.24 g/m
2
). The treatments were sublethal, as cricket mortal-
ities were low and evenly distributed among treatments and
controls. However, cricketsshowed significantly less mobility
and feeding behaviors with the high treatment, while both
treatments resulted in significantly lower weight and thorax
growth. The high treatment also increased the predation of
crickets by the spider, while spiders tended to move more
under such circumstances. However, and surprisingly, the
low treatment resulted in higher survival of the crickets than
in the controls. Overall, herbivory was reduced and predation
increased at sublethal concentrations of imidacloprid, suggest-
ing possible impacts through trophic interactions.
Significant changes in invertebrate community composition
were observed immediately after spray applications of fipronil
for locust (Chortoicetes terminifera) control in Queensland,
Australia, though the richness and abundance of species caught
in pan and pitfall traps were not significantly affected. The chang-
es in species composition for the flying insects (pan traps)
persisted for up to 79 days after spraying operations, whereas
those for the ground-dwelling invertebrates (pitfall traps) lasted
up to 189 days. The authors of this field study explained that a
long drought period that occurred during their 2-year monitoring
may have influenced the slow recovery of the invertebrate pop-
ulations (Walker et al. 2016). The composition of arthropod com-
munities was not significantly affected overtime in another study
that used fipronil sprays for locust control in New South Wales,
Australia (Maute et al. 2017a). However, springtails, mites, bee-
tles,crickets,psocopterans,anddipteransexperiencedshort-term
decreasesin abundance. The highest reductions were observed in
two ant species, one of which did not recover for longer than a
year. Because arthropod abundance and community assemblage
changedoverthe 2-year study,bothin control and treatment sites,
changes in patterns of local rainfall over the study period were
larger than changes in abundance due to pesticide treatment
(Maute et al. 2017a). The same authors found no impacts on
wood-eating termites activity, measured as consumption of
wooden baits, or species composition but such termites are soil
dwellers, are scarce in arid Australia and may not have been
exposed to the sprays (Maute et al. 2016). Equally, litter decom-
position carried out by soil microbial communities was not af-
fected by the fipronil sprays (Maute et al. 2017b).
The few species of earthworms used in toxicology tests are
more tolerant to neonicotinoids than other soil invertebrates.
However only few species have been studied and Eisenia
fetida, the species on which most studies were performed is
an epigean (i.e., surface-dwelling) compost worm and is not a
common species in forest or agricultural soil. The acute tox-
icities (LC50s in mg/kg soil) of five neonicotinoids to the
earthworm Eisenia fetida after 14 days exposure were deter-
mined as 4.34 for nitenpyram, 3.05 for imidacloprid, 2.69 for
acetamiprid, 2.68 for thiacloprid, and 0.93 for clothianidin
Tab l e 1 (continued)
Scientific name Taxon Acetamiprid Clothianidin Dinotefuran Imidacloprid Thiacloprid Thiamethoxam Fipronil References
Podisus maculiventris Hemiptera: Pentatomidae 4.7 De Cock et al. (1996)
Podisus maculiventris Hemiptera: Pentatomidae 5 Cutler et al. (2006)
Podisus nigrispinus Hemiptera: Pentatomidae 0.285 0.055 Torres and Ruberson (2004)
Chrysoperla rufilabris Neuroptera: Chrysopidae 121.7 Mizell and Sconyers (1992)
Scolothrips takahashii Thysanoptera: Thripidae 1.81 Mori and Gotoh (2001)
*μg/cm
2
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Tab l e 2 Lethal median concentrations (LC
50
, mg/L) of systemic pesticides to Hymenoptera parasitoids of crop pests
Scientific name Family Acetamiprid Clothianidin Dinotefuran Imidacloprid Nitenpyram Thiacloprid Thiamethoxam Fipronil References
Aphelinus mali Aphelinidae 0.16 (Cohen et al. 1996)
Aphytis melinus Aphelinidae 0.005 0.246 0.105 (Prabhaker et al. 2011;
Prabhaker et al. 2007)
Encarsia formosa Aphelinidae 12 0.98 0.397 Prabhaker et al. 2007 and 2011
Encarsia inaron Aphelinidae 208.9 (Sohrabi et al. 2013)
Eretmocerus eremicus Aphelinidae 108.3 1.93 1.01 Prabhaker et al. 2007 and 2011
Eretmocerus mundus Aphelinidae 4.75 Sohrabi et al. 2013
Apanteles subandinus Braconidae 530 (Symington and Horne 1998)
Aphidius colemani Braconidae 0.327 (Charles-Tollerup 2013)
Bracon hebetor Braconidae 0.082** (Danfa et al. 1998)
Cotesia chilonis Braconidae 0.0067 (Huang et al. 2011)
Cotesia vestalis Braconidae 0.475 (Wu and Jiang 2004)
Diaeretiella rapae Braconidae 5.1 0.4 (Wu et al. 2004)
Opius flavus Braconidae 0.059 (Wu et al. 2007)
Orgilus lepidus Braconidae 50 Symington and Horne 1998
Psyttalia concolor Braconidae ~ 150 < 40 (Adán et al. 2011)
Syngaster lepidus Braconidae 0.288 (Paine et al. 2011)
Haplogonatopus sp. Dryinidae 0.12 (Tanaka et al. 2000)
Avetianella longoi Encyrtidae 0.212 Paine et al. 2011
Copidosoma koehleri Encyrtidae 48 Symington and Horne 1998
Ooencyrtus nezarae Encyrtidae 50 (Alim and Lim 2014)
Neochrysocharis okazakii Eulophidae 0.0231 0.0035 (Tran and Ueno 2012)
Oomyzus sokolowskii Eulophidae 35.183 (Cordero et al. 2007)
Diadegma insulare Ichneumonoidae 41.5 (Hill and Foster 2000)
Diadegma insulare Ichneumonoidae 23.9 Cordero et al. 2007
Diadromus collaris Ichneumonoidae 0.12 Wu et al. 2007
Anagrus nilaparvatae Mymaridae 0.021 0.52 0.18 (Wang et al. 2008)
Anaphes iole Mymaridae 0.053** 1.7 (Williams III L et al. 2003)
Gonatocerus ashmeadi Mymaridae 0.134 2.63 1.44 Prabhaker et al. 2007 and 2011
Trissolcus nigripedius Platygastridae 500 (Lim and Mahmoud 2008)
Catolaccus grandis Pteromalidae 0.087 (Elzen et al. 1999)
Pteromalus puparum Pteromalidae 0.11 Wu et al. 2007
Spalangia endius Pteromalidae 52.2 * 17.92 * 41.94* (Burgess and King 2015)
Trichomalopsis sp. Pteromalidae 0.2 Wu et al. 2007
Urolepis rufipes Pteromalidae 0.8 * 10.* (Burgess and King 2016)
Gryon japonicum Scelionidae 500 Alim and Lim 2014
Haeckeliania sperata Trichogrammatidae 423 (Carrillo et al. 2009)
Trichogramma cacoeciae Trichogrammatidae 1.25 (Saber 2011)
Trichogramma chilonis Trichogrammatidae 0.0113 0.0027 0.0014 (Preetha et al. 2009)
Trichogramma chilonis Trichogrammatidae 0.376 (Wang et al. 2012a)
Trichogramma confusum Trichogrammatidae 93.2 754.2 0.84 176.5 0.24 0.86 (Wang et al. 2013)
Trichogramma evanescens Trichogrammatidae 24.46 50.28 2.9 17.24 1.12 (Wang et al. 2014)
Trichogramma japonicum Trichogrammatidae 25.39 95.48 75.26 0.4 0.92 (Zhao et al. 2012)
Trichogramma nubilale Trichogrammatidae 19.2 312 4.37 56.73 1.86 0.29 (Wang et al. 2012c)
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(Wang et al. 2015b). The authors reported that exposures in
the range 0.82.0 mg/kg also reduced the fecundity of this
species between 39.5 and 84% depending on the compounds,
while causing significant disruption of epidermal and midgut
tissues. The median number of hatched cocoons (EC50) for
earthworms exposed to imidacloprid was determined as
0.92 mg/kg soil, and its lowest observed effect concentrations
(LOECs) for hatchability, AChE activity, growth, and DNA
damage were 0.02, 0.1, 0.5, and 0.5 mg/kg soil, respectively
(Wang et al. 2015c). Mixtures of imidacloprid and lambda-
cyhalotrin appear to have antagonistic toxicity in these earth-
worms (Wang et al. 2015f). Other authors have demonstrated
the low toxicity of the new compound guadipyr to Eisenia
fetida, as concentrations of this insecticide in soil below
100 mg/kg in did not affect the growth or the reproduction
output. Only increases in enzymatic activities of superoxidase
dismutase and catalase were observed in the first few days of
exposure, returning to normal levels afterwards (Wang et al.
2015a). The 48-h LC50 for acute toxicity of nitenpyram to the
earthworm Pheretima posthuma was determined at 0.29 mg/
kg soil (Hussain et al. 2017).
A study of the nematode communities in soils of corn fields
treated with clothianidin, either as granules or in seed-coating,
found significant differences in species richness and diversity
compared to untreated control fields on two separate years. The
main driver in community composition of nematodes was the
year and month of sampling, but clothianidin treatments reduced
thediversity of species significantly,with up to 5 species outof36
being absent, even if total abundances between treatments and
controls were statistically similar (Čerevková et al. 2017).
The enantiomers of fipronil appear to have different toxic-
ity to the earthworm Eisenia fetida, with S-fipronil having a
higher subchronic toxicity and bioaccumulation potential than
R-fipronil (Qin et al. 2015). Weight reductions after 28 days
exposure to concentrations in soil ranging 501000 mg/kg
were 2353% for the R-fipronil and 3862% for the S-
fipronil enantiomers. Residue accumulation in the earthworms
reached a peak after 10 days exposure and then declined,
following the dissipation pattern of the initial fipronil race-
mate in the soil. In tissues, fipronil, fipronil sulfone, and
fipronil sulfide were detected, with bioaccumulation factors
of 0.50.75 and a biological half-life in the range 1.52.1 days
(Qin et al. 2015).
Studies on the effect of systemic pesticides on soil organ-
isms are limited to a few species and the chosen species may
not be the most ecologically meaningful. Therefore, the true
impact of systemic pesticides on soil organisms and associated
functions remains an important knowledge gap.
Effects on aquatic invertebrates
A comprehensive review of the acute and chronic toxicity of
neonicotinoids to 49 species of aquatic insects and
Tab l e 2 (continued)
Scientific name Family Acetamiprid Clothianidin Dinotefuran Imidacloprid Nitenpyram Thiacloprid Thiamethoxam Fipronil References
Trichogramma nubilale Trichogrammatidae 0.609 2 (Chen et al. 2013)
Trichogramma ostriniae Trichogrammatidae 0.12 2.94 1.58 0.14 (Li et al. 2015b)
Trichogramma ostriniae Trichogrammatidae 43.02 503.6 4.93 376.3 2.48 0.14 (Wang et al. 2012b)
Trichogramma pretiosum Trichogrammatidae 0.53 § (Williams III L and Price 2004)
*ng/cm
2
**kg/ha
§
LC90
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crustaceans, spanning 12 invertebrate orders, indicates that
differences in sensitivity among aquatic invertebrate species
range several orders of magnitude (Morrissey et al. 2015).
More than two thirds of the data refer to imidacloprid, for
which acute LC50s range from 4 μg/L in the most susceptible
insect orders (Ephemeroptera, Trichoptera, and Diptera) to
values exceeding 44,000 μg/L in the most tolerant cladoceran
crustaceans. It is unfortunate that the standard species used in
regulatory assessments, namely Daphnia magna,hasatypical
LC50 of about 100,000 μg/L, and this caused regulators to
underestimate risks for some time. The study recommended
ecological thresholds for neonicotinoids in water at concentra-
tions below 0.2 μg/L for short-term acute exposures and
0.035 μg/L for long-term chronic exposures to avoid ecolog-
ical impacts on aquatic invertebrate communities (Morrissey
et al. 2015). Some of these impacts were described in a previ-
ous review (Pisa et al. 2015), and links between the toxic
effects at the individual species, populations, and communities
levels with impacts on aquatic and terrestrial ecosystems have
been published more recently (Sánchez-Bayo et al. 2016a).
The decline of dragonflies and damselflies (Odonata) in
Japan since the early 1990s has been blamed on the introduc-
tion of systemic insecticides in that country (Jinguji and Uéda
2015), but solid evidence was lacking. Impacts of these insec-
ticides applied to rice seedlings in nursery-boxes at recom-
mended rates were tested on the dragonfly Sympetrum
frequens: none of the 50 nymphs introduced in experimental
lysimeters treated with either imidacloprid or fipronil survived
after 1 month, and only 13% remained in those treated with
dinotefuran. Although the rate of adult emergence in the
dinotefuran treatment was similar to that in the controls, the
average head width of the dragonflies in this treatment was
significantly narrower (Jinguji and Uéda 2015). In a similar
study usingpaddy mesocosms treated with either clothianidin,
fipronil, or chlorantraniliprole, the abundance of Odonata spe-
cies in the clothianidin and specially in the fipronil-treated
paddies was very low compared to controls and the other
treatment (Kasai et al. 2016). Plankton species also declined
in the clothianidin and chlorantraniliprole treatments right af-
ter the applications, but they recovered when their initial con-
centrations decreased to minimal levels. Previous mesocosm
studies in Japan had showed the toxicity of imidacloprid to
Odonata species when applied at the recommended rates to
rice paddies (10 kg/ha). However, the study by Kobashi et al.
(2017) also showed compensation among the predatory in-
sects: while populations of Crocothemis servilia mariannae
and Lyriothemis pachygastra nymphs were significantly re-
duced, those of Orthetrum albistylum speciosum increased
slightly throughout the 5-month experimental period. Large
decreases in the abundance of a common predator,
Notonecta triguttata, were also observed, and the Guignotus
japonicus disappeared, the effects on both species resulting
from a delayed but measurable chronic toxicity (Kobashi
et al. 2017). Other authors have determined the 48-h LC50
for clothianidin in North American dragonflies in the range
865 to 1245 μg/L (Miles et al. 2017).
Mayflies (Ephemeroptera) comprise other insect taxa very
susceptible to neonicotinoids. The acute and chronic toxicity
(28 days exposure) of thiamethoxam, thiacloprid, and
imidacloprid to Cloeon dipterum, and their seasonal variability
was studied by van den Brink et al. (2016). Thiacloprid was twice
as toxic to the winter generation as the other two neonicotinoids,
whereas both acute and chronic toxicity of imidacloprid to the
summer generation was much higher than to the winter one.
Camp and Buchwalter (2016) demonstrated that the higher sus-
ceptibility of 6 species of aquatic insects to imidacloprid during
summer is due to the higher water temperatures during that sea-
son: the time-to-effect for sublethal impairment and immobility
was significantly decreased with increasing temperature from 15
to 25 °C because the intake of the toxicant and metabolism also
increased accordingly. For Cloeon dipterum, lethal median con-
centrations (LC50s) of the studied neonicotinoids
(thiamethoxam, thiacloprid, and imidacloprid) decreased by a
factorof3to6timesbetween24and96hofexposureineither
season (van den Brink et al. 2016).Thesameresultwasfound
with the lotic mayfly Isonychia bicolor exposedtoimidacloprid
(Camp and Buchwalter 2016). Moreover, chronic exposures of
C. dipterum resulted in LC50s of 0.30 μg/L for thiacloprid,
0.32 μg/L for imidacloprid and 0.8 μg/L for thiamethoxam, the
latter LC50s being 270, 800 and 100 times lower than their re-
spectiveonesat24h(vandenBrinketal.2016). Also, LC50s for
acute exposure of the freshwater amphipod Gammarus
kischineffensis to thiamethoxam dropped from 75.6 μg/L at
24hto3.7μg/L at 96 h, that is a 20-fold decrease in concentration
in 4 days to achieve the same mortality effect (Uğurlu et al. 2015).
These studies confirm the delayed and extreme chronic toxicity
of neonicotinoids to aquatic organisms.
New toxicity data of aquatic predatory insects are now avail-
able for clothianidin (Miles et al. 2017).The48-hLC50forthe
aquatic beetle Graphoderus fascicollis (Dytiscidae) was deter-
mined at 2 μg/L, which indicates the susceptibility of this species
compared to that of four species of water bugs (LC50 range 56
805 μg/L) and three species of dragonflies (LC50 range 865
1245 μg/L). The water bug Belostoma flumineum displayed a
dose-dependent reduction in feeding rate after exposure to sub-
lethal concentrations of clothianidin. The authors also carried out
amesocosmstudy to investigate the effect of three concentrations
of clothianidin (0.6, 5, and 352 μg/L) in the arthropod commu-
nities. Predatory invertebrates experienced significant mortality
with increasing levels of the insecticide in water, concomitant
with increases of their prey up to 50% at the highest concentra-
tion, indicating a top-down trophic cascade in community abun-
dance (Miles et al. 2017).
In laboratory tests with larvae of Chironomus dilutus,the14-
day LC50s for imidacloprid, clothianidin, and thiamethoxam
were 1.52, 2.41, and 23.60 μg/L, respectively. However, the
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40-d EC50s for adult emergence under exposure to the same
chemicals were 0.39, 0.28, and 4.13 μg/L, respectively. This
indicates that sublethal concentrations that prevent emergence
of this key wetland species are between4and9timeslowerthan
those that cause mortality to the larvae (Cavallaro et al. 2017).
Exposure of Chironomus riparius larvae to various mixtures of
pyrethroid (deltamethrin and esfenvalerate) and neonicotinoid
insecticides (imidacloprid and thiacloprid) at 50% oftheir known
LC50s showed sometimes additive and other times antagonistic
effects on survival (Kunce et al. 2015). In the case of the amphi-
pod Hyalella azteca, combined exposure to imidacloprid and
cyfluthrin resulted in mortality ratios of 1.7 to 2.7 higher than
either insecticide alone, indicating greater than additive toxicity
(Lanteigne et al. 2015).
Available toxicity data for amphipods indicate that these
detritivores of organic material are less susceptible to
neonicotinoids than insect larvae by one order of magnitude or
more (Morrissey et al. 2015). However, such differences tend to
be species specific. For example, recent studies have shown that
the amphipod Gammarus fossarum is more susceptible than the
caddisfly Chaetopteryx villosa when exposed to three
neonicotinoids (imidacloprid, thiacloprid, and acetamiprid), ei-
ther alone or in mixtures (Englert et al. 2017). Furthermore, the
same study found that combined exposure of these shredder spe-
cies to neonicotinoid residues in water and in food (tree leaves)
had more negative impacts on their survival than direct exposure
to contaminated water alone. Exposure of benthic organisms to
residual neonicotinoids in water is already widespread in Europe:
47%ofthe19amphipods(Dikerogammarusspp.) collectedfrom
the Danube river in eastern Germany had residues of thiacloprid
at levels 0.10.39 ppb (wet body weight) (Inostroza et al. 2016).
Mollusks are known to be quite tolerant of neonicotinoids
(Morrissey et al. 2015), with water snails (Physa acuta and
Helisoma trivolvis) having no mortality after exposure to
327 mg/L clothianidin for 2 days (Miles et al. 2017). It appears
that the mechanism of toxicity of these insecticides on these
organisms is different, as neonicotinoids inhibit the nACh
receptors in the pond snail (Lymnaea stagnalis) instead of
acting agonistically (Vehovszky et al. 2015). At environmen-
tally relevant concentrations (0.1 to 100 μg/L), imidacloprid
downregulated the production of many fatty acids in the
snails, while the levels of polyamines, spermidine and putres-
cine increased, indicating neuron cell injury. Cholinergic gene
expression was also increased as the snails tried to overcome
imidacloprid binding to the nAChRs (Tufi et al. 2015).
Exposure of the snails to mixtures of neonicotinoids and other
pesticides found in water from agricultural areas in the
Netherlands showed more disturbed metabolic pathways than
exposures to individual chemicals (Tufi et al. 2016). Toxicity
tests of a suite of neonicotinoids to the Ramshorn snail
(Planorbella pilsbryi)showed7-dayLC50sof4000 μg/L
for imidacloprid,