BioRisk 8: 39-5 | (20 | 3) a ry sneer ae
doi: 10.3897/biorisk.8.325 | RESEARCH ARTICLE &% B | O R IS k

www.pensoftonline.net/biorisk

Anuran amphibians as indicators of changes in
aquatic and terrestrial ecosystems following
GM crop cultivation: a monitoring guideline

Susanne Bdll', Benedikt R. Schmidt’, Michael Veith?, Norman Wagner’, Dennis
Rédder*, Cathrin Weimann?, Tom Kirschey°, Stefan Létters?

| Agency for Population Ecology and Nature Conservancy, In der Setg 10, 97218 Gerbrunn, Germany
2 KARCH, Passage Maximilien-de-Meuron 6, 2000 Neuchatel, Switzerland and Institut fur Evolutionsbio-
logie und Umweltwissenschaften, Universitat Ziirich, Winterthurerstrasse 190, 8057 Ziirich, Switzerland 3
Trier University, Biogeography Department, 54286 Trier, Germany 4 Zoologisches Forschungsmuseum Konig,
Adenauerallee 160, 53113 Bonn, Germany 5 Naturschutzbund Deutschland, NABU Brandenburg, Linden-
str.34, 144467 Potsdam, Germany

Corresponding author: Susanne Boll (susanneboell@gmail.com)

Academic editor: /. Settele | Received 18 April 2012 | Accepted 1 February 2013 | Published 8 August 2013

Citation: Boll S, Schmidt BR, Veith M, Wagner N, Rodder D, Weimann C, Kirschey T, Létters S$ (2013) Anuran
amphibians as indicators of changes in aquatic and terrestrial ecosystems following GM crop cultivation: a monitoring

guideline. BioRisk 8: 39-51. doi: 10.3897/biorisk.8.3251

Abstract

Amphibians are a suitable indicator group for monitoring possible negative direct or indirect effects of
GMO cultivation at the individual and population level. Direct effects could occur in aquatic ecosystems
via uptake of GM pollen or GM detritus by anuran larvae. However, indirect negative effects caused by
changes in cultivation practices (changes in pesticide use, for instance) are more likely. The VDI Guide-
line 4333 aims to ensure comprehensive monitoring of the different life-stages of anuran species that are
common in agricultural landscapes of Austria, Germany and Switzerland. The guideline includes a novel
approach to tadpole monitoring. To assess immediate effects, tadpole, metamorph and adult deforma-
tion rates are compared with naturally occurring deformation rates. Adult population size, adult body
condition and juvenile emergence are monitored over multiple years to assess long-term effects of GM
crop cultivation on population viability. At each study site, monitoring has to be carried out at multiple
amphibian breeding sites which differ in their exposure to GM crop cultivation. All monitoring data have
to be stored in a central database for future meta-analyses. This will ultimately allow for generalized state-
ments about the impact of GM crop cultivation on amphibians. Although specifically designed for GM
crops, VDI Guideline 4333 may also serve as a model for studying the effects of a wider range of stressors

on amphibian populations in agriculture and forestry.

Copyright Susanne Boll et al. This is an open access article distributed under the terms of the Creative Commons Attribution License 3.0
(CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

40 Susanne Boll et al. / BioRisk 8: 39-51 (2013)

Keywords

GMO, monitoring guideline, agriculture, pesticide, deformation, population size, tadpole, recruitment, adult

Introduction

EU Directive 2001/18/EC on the deliberate release of genetically modified organisms
(GMO) into the environment requires the assessment of environmental impacts of
GMO including direct, indirect, immediate and delayed effects on the environment,
further specified in Council Decision 2002/811/EC. In the case of transgenic crops
resistant to herbicides, this means that besides evaluating the environmental impacts
of the genetically modified plant itself, the environmental impacts of specific herbicide
programmes and altered agricultural practices associated with this crop have to be as-
sessed. GM crops may affect not only terrestrial, but also adjacent aquatic ecosystems
such as ponds, ditches or run-off water bodies (Mann et al. 2009).

Amphibians are considered a particularly sensitive indicator group for environ-
mental stress in aquatic and terrestrial ecosystems (e.g. Blaustein and Johnson 2003;
Blaustein and Kats 2003). Owing to their complex life cycle, the most sensitive stages
of embryonic and larval development are usually spent in aquatic habitats, while most
juveniles and adults live in terrestrial habitats. Furthermore, as amphibians depend on
water for breeding, adults of many species, especially in temperate regions, show exten-
sive annual migrations between winter, breeding, and summer habitats.

As tadpoles of frogs and toads (anurans) are predominantly herbivores, they should
be more sensitive to the entry of GMO products into aquatic systems than carnivorous
newt and salamander larvae. Food uptake of GMO pollen, GMO detritus, or GMO-
contaminated sediment could directly affect larval health, growth and development.
However, indirect effects are expected to have much greater impacts, since GMO cul-
tivation is likely to change the way farmers use pesticides, both in terms of cultivation
practices and pesticide application regimes and doses. ‘This may lead to significant effects,
such as deformities and increased mortality as observed in anuran larvae (Mann et al.
2009; Paganelli et al. 2010; Relyea 2005b). While interactions between toxic effects and
other ecological factors can amplify lethal effects of pesticides (Relyea and Mills 2001),
even sublethal effects on larval growth and development can impair amphibian popula-
tions permanently (Jones et al. 2010; Relyea 2005a; Relyea 2005b). In agricultural sys-
tems, amphibians may be particularly susceptible to the toxic effects of pesticides because
their breeding sites are often shallow ponds or temporary pools that may accumulate pol-
lutants without substantial dilution (Howe et al. 2004; Mann et al. 2003). Furthermore,
depending on application times and frequencies, lethal damage of amphibians during
migrations in their terrestrial habitat is to be expected (Briihl et al. 2011).

Anurans are not only sensitive bioindicators for environmental change, they are
also well suited for monitoring programmes due to:

e the substantial state of knowledge concerning the biology, ecology and conserva-
tion status of the different species,

Anuran amphibians as indicators of changes in aquatic and terrestrial ecosystems... 4]

e their predictability at the breeding site, and
e the fact that breeding, larval development and migrations occur in spring and
summer, the season of pesticide applications.

Here, we summarize the possible negative impact of direct and indirect effects of
GM crop cultivation on amphibians. We describe the study design of VDI Guideline
4333 that allows assessment of these effects using a standard protocol. ‘This guideline
deals only with anuran amphibians, as they are easier and more reliably to survey than
salamanders and newts. Besides, anuran larvae should be more sensitive to direct ef-
fects of GMO products due to their herbivorous feeding behaviour.

Direct potential effects of GMOs on anuran amphibians

Direct effects of GMO products on amphibians have not yet been studied. However,
effects of GM crops could result from the accumulation of detritus or pollen in the
aquatic environment. Many tadpoles are non-discriminatory feeders and ingest high
amounts of detritus and pollen (e.g. Wagner 1986). Although toxins in GM crops
(e.g. Bt toxin) should not have an impact on tadpoles, such effects cannot be ruled
out. Additionally, future GM crops may contain toxins that may have direct negative
effects on amphibians.

Indirect potential effects of GMOs on anuran amphibians

Today, nearly all GM crops have a genetically engineered resistance to non-selective her-
bicides (ISAAA: Global Status of Commercialized Biotech/GM Crops: 2009. The first
fourteen years, 1996 to 2009. http://www.isaaa.org/resources/publications/briefs/41/
executivesummary/default.asp), mostly based on the active ingredient glyphosate
(Duke and Powles 2008). The use of herbicides associated with the cultivation of GM
crops may therefore indirectly affect amphibians.

Amphibians may be exposed to these pesticides in many ways. Frogs and toads
may become contaminated; for instance, migrating terrestrial amphibians (adults, ju-
veniles or metamorphs) can be over-sprayed or can get in contact with pesticides ad-
hering to soil or plant material (Briihl et al. 2011). Pesticides may also contaminate
amphibians via the food chain, when amphibians prey on insects that have come into
contact with pesticides (McComb et al. 2008). Anuran eggs and larvae can be affected
through the accumulation of pesticides or their degradation products in aquatic eco-
systems via wind drift, drainage or run-off. In agricultural systems, amphibians may be
particularly susceptible to the toxic effects of pesticides because their breeding sites are
often shallow ponds or temporary pools that may contain higher concentrations than
larger water bodies (Mann et al. 2003; Howe et al. 2004).

Within recent decades, the number of herbicide-resistant weeds has increased
dramatically due to unsustainable weed management with the almost exclusive use

42 Susanne Boll et al. / BioRisk 8: 39-51 (2013)

of only one active ingredient (Heap 2011: The International Survey of Herbicide-
Resistant Weeds. http://www.weedscience.org/ChronIncrease.gif). This is particular-
ly true for herbicide-resistant GM crop cultivations (Powles 2008). Weed resistance
leads to higher doses and additional applications of herbicides (Schiitte and Mertens
2010). Furthermore, non-selective herbicides can be applied later in the year than
selective herbicides (BVL 2010). Hence, the exposure risk for different amphibian
species and different life-stages may shift. Applications later in the year may include
an increased spraying height that can lead to higher aerial pesticide drift. The likeli-
hood of heavy rainfall events in early summer is increasing in Western Europe due to
climate change. Increased drift, heavier rainfall and greater erosion may cause adja-
cent aquatic habitats to become more heavily contaminated through the use of non-
selective herbicides (SRU 2008).

Pesticides may lead to a reduction in insect abundance, and subsequently to re-
duced food availability for amphibians. Direct contact of amphibians with pesticides
is known to have lethal and sublethal effects. These include increased mortality due to
acute toxicity (Howe et al. 1998, Relyea 2005b) or teratogenic (Paganelli et al. 2010)
and genotoxic effects (Clements et al. 1997).

Tadpoles often show pesticide-induced deformations in body and tail shape (e.g.
Cooke 1981, Lajmanovich et al. 2003). Some pesticides are also endocrine disruptors
that potentially target the thyroid axis (Howe et al. 2004), leading to an imbalance in
the sex ratio of the population (Hayes et al. 2006). They are also known to have an
immunosuppressive effect (Rohr et al. 2008). In addition, pesticides may interact with
other stressors such as predators (Relyea and Mills 2001, Boone and James 2003). All
this applies to many pesticides, but some non-selective herbicides seem to be amongst
the most dangerous for amphibians (see Williams and Semlitsch 2010).

VDI Guideline 4333: an anuran monitoring scheme to assess possible ad-
verse effects of GMO cultivation on aquatic and terrestrial environments

The “VDI Society Technologies of Life Sciences” prepared a guideline on how to assess the
potential negative effects of GM crop cultivation on amphibians, namely anurans, occur-
ring in Austria, Germany and Switzerland. It is published as VDI Guideline 4333 "Moni-
toring der Wirkungen des Anbaus von gentechnisch veranderten Organismen (GVO) —
Standardisierte Erfassung von Amphibien/Monitoring the effects of genetically modified
organisms (GMOs) — Standardized monitoring of amphibians". Here we summarize the
essential elements of this guideline; for details we refer the reader to the guideline itself
(VDI 2013; www.vdi.de/4333).

VDI Guideline 4333 describes a study design that can be used to assess (i) immediate
negative impacts, and (ii) long-term effects on anuran populations. To assess immediate
effects, tadpole, metamorphic and adult deformation rates need to be recorded. Increases
in these rates are common responses to herbicides (e.g. Cooke 1981, Lajmanovich et al.
2003, Paganelli et al. 2010). Herbicides can directly but also indirectly cause deformities

Anuran amphibians as indicators of changes in aquatic and terrestrial ecosystems... 43

by an enhanced susceptibility to trematode infection (Kiesecker 2002, Rohr et al. 2008).
However, deformations, even when occurring at high rates, may not substantially affect
population viability if density dependence is high (e.g. Forbes and Calow 2002; Veith
and Viertel 1993). Thus, to assess long-term effects, the guideline stipulates studying
changes in population size in order to track possible population declines.

Standard methods for monitoring adult populations of amphibians are well estab-
lished (e.g. Dodd 2010; Heyer et al. 1994). However, tadpole monitoring is a rather
new approach. Therefore, the VDI Guideline 4333 aims to set standards for assessing
the immediate negative impacts of GM crop cultivation and pesticides on aquatic
habitats using anuran larvae.

A first crucial step in monitoring is to define the study area and duration of
the monitoring programme. Due to natural fluctuations in amphibian population
parameters, potential negative impacts on amphibian populations in habitats influ-
enced by GM crop cultivation should be controlled by comparison with unaffected
habitats. However, since it is next to impossible to identify potentially unaffected
sites a priori, VDI guideline 4333 defines a set of water bodies with different magni-
tudes of exposure, which are to be monitored per GMO cultivation site. In addition
to the three most exposed breeding habitats, three additional, less exposed breeding
habitats within a radius of up to 1000 m from the margins of the GMO field will be
selected for monitoring (Fig. 1A). The expected magnitude of exposure is quantified
via GIS on the basis of the distance between the water body and the GMO field, the
prevailing relief (topography) and the water flow regime. ‘This spatial set-up allows
an assessment of possible negative effects along a gradient of exposure intensity.
For each of these six monitoring sites, the expected magnitude of exposure will be
quantified via GIS analysis: buffers with different radii are drawn around each site,
and within the buffers the proportion of land used for GMO cultivation as well as
the ratios of general land use (arable land, grassland, woodland, residential area) are
determined (Fig. 1B).

To account for population trends, the monitoring has to be conducted during the
period of GMO cultivation and should be continued for at least two times the generation
time of a species after cultivation has ceased. Since generation time varies across species and
may also be habitat-specific, the guideline suggests this extended period to last for ten years.

To minimize the impact of monitoring on the respective amphibian populations
and to increase comparability across monitoring studies, VDI Guideline 4333 recom-
mends focusing on species occurring in the agrarian landscape that are widely distrib-
uted throughout Austria, Germany and Switzerland. It therefore distinguishes between
‘obligate’ and ‘supplementary’ species. Obligate species, namely the Common toad,
Bufo bufo, and the Common frog, Rana temporaria, are widely distributed in Western
Europe and, as indicated by their vernacular names, are common in most areas and
sites. Where one or both of these occur in sufficient numbers, they must be included
in the monitoring. However, since amphibian species differ in their susceptibility to
pesticides (e.g. Cooke 1981, Kerby et al. 2010), it is advisable to study additional spe-
cies wherever possible. Therefore, supplementary species should be added where they

44 Susanne Boll et al. / BioRisk 8: 39-51 (2013)

A

es a ae ae eK eK ee ee ee ee ee ee ee Kee

7’ study area @ @
O © @

CD Area under GMO cultivation
ei) Selected pond

\ O OQ’ © Non-selected pond

]
]
]
]
]
Legend
]
]
]
I
|

Sal See 2 oe i SS ES Se eat Oe EE 1000m radius around GMO cultivation
Exposure gradient
Fi a Ai a a ha RR ie aca mae B
/ ‘N
f \
3 e® ©
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rete |
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| van Gb) ee fae
oA ©, “fe, () area under GMO cultivation

j © Selected pond
ce am eee @ Pond under consideration
eS Non-selected pond

aatbeski Different radii around the pond
under consideration

Figure |. Spatial monitoring design of amphibian populations to quantify possible adverse effects of
GMO cultivation. A Selection of an array of study ponds with different exposure probabilities B Quan-
tifying the proportion of GMO cultivation within different radii around each study pond (in accordance
with VDI guideline 4333).

are regionally or locally as common as or even more prevalent than obligate species.
Supplementary species may be typical for substantial parts of the area of influence of
the VDI guideline. They can therefore replace obligate species where one or both of
them are not present in sufficient numbers.

The monitoring guideline stipulates investigating the following life-stages:

(i) | Reproducing adults: In an initial step, information about the reproductive popu-
lation in a given year and its changes over time has to be recorded. All males and

Anuran amphibians as indicators of changes in aquatic and terrestrial ecosystems... 45

females migrating in spring to a breeding site are counted using drift fences with
pit fall traps (e.g. Dodd 2010; Hachtel et al. 2009; Henle and Veith 2004). Be-
sides identifying species and sex, all specimens are examined for abnormalities.
Information on individual body condition, which is inferred from measurements
of body length and body mass, may provide further insights into long-term ef-
fects of GM crop cultivation on population viability. The counted number of
amphibians is only a reliable index of population size if detection probabilities
are high (Bailey et al. 2004; Schmidt and Pellet 2009). Therefore, the drift fence
must be operated very carefully to ensure that detection probabilities are high
and show low variability.

(ii) | Tadpoles: Deformations in body and tail shape or of extremities are naturally ob-
served at a rate of less than 5% in most anuran larvae (Cooke 1981; Mann et al.
2009; Piha et al. 2006). Monitoring tadpoles during early and late development
will therefore allow the detection of unusual increases of deformation rates.

(iii) Metamorphs: Metamorphs are counted using drift fences with pit fall traps.
They are checked for abnormalities in the same way as adults. Comparison of
quantitative data on juvenile emergence across ponds along the exposure gradi-
ent and across years provides the opportunity to estimate the effect of GMO
cultivation on the annual recruitment of a population. In the case of an appar-
ent mass mortality, VDI Guideline 4333 recommends preserving metamorphs
for subsequent histological examination.

If negative effects are detected in tadpoles or metamorphs, detailed water and sedi-
ment analyses have to be carried out in the respective water body in subsequent years.
These along with the accompanying monitoring data will show during the follow-
ing years if negative effects are related to GM crop cultivation. Such supplementary
analyses should focus on transgenes, GMO by-products and pesticides using available
standard procedures (EC 2009; EC 2010; Ziighart and Dorpinghaus 2004).

The results for each monitoring must be stored in a central database for meta-
analyses. Ultimately, this will allow for generalized statements about the impact of GM
crop cultivation on anurans across different species, habitats and exposure intensities.

Discussion

Agricultural practices are known to affect biodiversity (Ellis et al. 2010; Jenkins 2003).
The use of GMOs in agriculture is likely to affect biodiversity as well. Here we describe
how to assess the impact of GM crop cultivation on amphibian populations. Our as-
sumption is that GM crops probably have no direct negative effects on amphibians,
whereas indirect negative effects are to be expected. These may be caused by changes in
pesticide use that are typically associated with the cultivation of GM crops.

Pesticides are well known to affect amphibians (Bridges 1997; Brunelli et al. 2010;
Davidson 2004, Howe et al. 2004; Paganelli et al. 2010; Relyea 2005a; Relyea 2005b;

46 Susanne Boll et al. / BioRisk 8: 39-51 (2013)

Relyea et al. 2005). Though effects on individuals do not necessarily impair population
viability (Forbes and Calow 2002; Rohr et al. 2006; Schmidt 2004), an ultimate deteri-
oration cannot be excluded. A comprehensive assessment of the impact of GM crop cul-
tivation on amphibians that includes short and long-term effects is therefore necessary.

VDI Guideline 4333 is designed to assess the potential impact of GM crop cultiva-
tion on amphibian populations in an agricultural landscape. However, it can also serve
as a general guideline that can be used to study the effects of a wide range of chemi-
cal stressors (pesticides, fertilizers, endocrine-disrupting chemicals, etc.) on amphibian
populations in agriculture or forestry. The guideline describes how to quantify negative
effects on amphibians during their aquatic and terrestrial life-stages. This comprehen-
sive approach is novel as it includes the most sensitive stages of larval development in
their aquatic environment.

Quantifying the possible effects of GM crop cultivation on natural amphibian pop-
ulations is challenging. A variety of effects may occur that have to be considered and
measured in the field. To design an operational monitoring scheme, we decided to focus
on four response variables that can easily be measured in the field: (i) rate of deformations
in the larval, juvenile and adult life-stages, (ii) number of metamorphs, (iii) number of
breeding adults, and (iv) body condition of breeding adults. We excluded further poten-
tial response variables (e.g. histological analyses of damaged tissues, skeletochronology to
determine shifts in adult age structure due to massive die-offs during pre-reproductive
stages, capture-mark-recapture estimates of survival and detection probabilities, struc-
tural changes in the agricultural landscape associated with GM crop cultivation) primar-
ily because they would be too difficult to be quantified on a large-scale. Nevertheless, we
are confident that the chosen response variables are adequate to detect potential effects
of the cultivation of GM crops on individual and population levels.

Deformities of tadpoles and body condition of adults quantify effects on individu-
als, while adult population size and the number of metamorphs (emergence) measure
effects at the population level. Individual-level effects may be viewed as precursors to
future negative population effects. Reading (2007) showed that there was a positive
correlation between adult survival and body condition. Emergence is a crucial compo-
nent of recruitment and thus a major driving force of amphibian population dynamics
(Hels and Nachman 2002; Lampo and De Leo 1998; Schmidt 2011). A decline in
abundance is a population-level effect which indicates either that the population was
unable to compensate a prior negative effect on individuals (e.g. increased mortality in
the tadpole stage) or that post-exposure effects may have occurred (Forbes and Calow
2002; Rohr et al. 2006). Because some effects may only become apparent years after
exposure, it is important to continue studying the possible consequences of GM crop
cultivation after cultivation has ended.

To assess the impact of GM crop cultivation on amphibians, their populations
must be studied at multiple ponds. As “control ponds” are hardly found at study sites,
in VDI Guideline 4333 a GIS-based approach was taken to determine how much each
pond may be affected by GM crops. Using circular buffers with varying radii around the
ponds and determining the relative extent of GM crop within these buffers, ponds can

Anuran amphibians as indicators of changes in aquatic and terrestrial ecosystems... 47

be arranged along a gradient of exposure to GM crop cultivation. We expect that this
approach along with a subsequent meta-analysis of all studied GMO sites will allow a
fairly precise determination of how GM crop cultivation affects amphibian populations.

The approach described in VDI Guideline 4333 to quantifying the effects of cul-
tivation of GM crops on amphibians is correlative. Therefore, it cannot prove that any
observed alteration was caused by either direct or indirect effects of GM crop cultiva-
tion. Experiments that could prove causality at population level would in principle
be possible (see Krebs et al. (1995) and Hudson et al. (1998) for case studies where
population dynamics were experimentally manipulated). However, they would be ex-
tremely laborious and, at the very least, in most, if not all, cases unethical.

The proposed study design can be viewed as a pseudo-experiment, with each cul-
tivation of a GM crop in the vicinity of amphibian breeding sites being a different
and independent experimental treatment. If, in fact, effects are observed, it is then
necessary to conduct experiments to establish cause-effect relationships. Deformities,
tadpole survival or adult body condition may also be response variables in experi-
mental studies. A combination of field studies, experiments and population modelling
will certainly give the most compelling evidence as to whether the cultivation of GM
crops negatively affects amphibian population dynamics. EU legislation requires that
the potential effects of the cultivation of GM crops are assessed whenever and wher-
ever GM crops are used. The data from all these studies need to be stored in a central
database, so that it will be possible to conduct a meta-analysis at a later stage. Such a
meta-analysis based on a large number of studies would gain a high statistical power
and would therefore allow a much greater understanding of the effects of GM crop
cultivation on amphibians.

Conclusions

Monitoring the effects of GM crops on biodiversity is an important and challeng-
ing task. Guidelines for standard monitoring, such as VDI Guideline 4333, are an
important step towards this direction. Importantly, the methodology described in the
guideline and in this article is general and could also be used to design an observational
study to assess the effects of other anthropogenic stressors on amphibians.

Acknowledgments

We are grateful to Heike Beismann and Martin Follmann of VDI Society Technologies
of Life Sciences, Wiebke Ziighart (Federal Agency for Nature Conservation, Germany)
and Andreas Lang (Institute of Environmental Geosciences, University of Basel) for
support when writing VDI Guideline 4333 and for the offer to write this paper. We
would also like to thank two anonymous reviewers for their constructive comments on
an earlier version of the manuscript.

48 Susanne Boll et al. / BioRisk 8: 39-51 (2013)

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