Science of the Total Environment 660 (2019) 126–135

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Do agricultural pesticides in streams influence riparian spiders?

Nadin Graf a,⁎, Karina P. Battes b, Mirela Cimpean b, Pitt Dittrich a, Martin H. Entling a, Moritz Link a,
Andreas Scharmüller a, Verena C. Schreiner a, Eduard Szöcs a, Ralf B. Schäfer a
a
Institue for Environmental Sciences, University Koblenz-Landau, Fortstraße 7, 76829 Landau, Germany
b
Department of Taxonomy and Ecology, Faculty of Biology and Geology, Babeș-Bolyai University, 5-7 Clinicilor Str., 400006 Cluj-Napoca, Romania

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Pesticide toxicity and shading are main

drivers of riparian spider community
composition.
• Spider abundance and richness are neg-
atively correlated with increasing toxic-
ity.
• Traits are associated with land use and
local environmental conditions

a r t i c l e i n f o a b s t r a c t

Article history: Freshwater ecosystems are coupled with their riparian area. Emerging insects are prey for predators in the ripar-
Received 31 August 2018 ian zone, enriching the terrestrial ecosystem with energy and nutrients. Stressors associated with agriculture can
Received in revised form 21 December 2018 alter insect communities in water and on land, resulting in complex response patterns of terrestrial predators re-
Accepted 24 December 2018
lying on prey from both systems. Examining the effects from individual agricultural stressors such as pesticides is
Available online 6 January 2019
hampered in landscapes with intensive agriculture where multiple stressors like habitat degradation and typi-
Editor: Henner Hollert cally co-occur. In rural regions of Eastern Europe, traditional low intensity agriculture based on working animals
and human labour prevails alongside intensive, mechanised agriculture. Assuming that low-intensity agriculture
Keywords: relies on no or limited pesticide use, whereas fertilizer use is similar across different agricultural intensities, such
Biodiversity regions may allow to study in-stream pesticide effects independent from other stressors, such as nutrient input or
Araneae habitat degradation. We examined the taxonomic and trait response of riparian spider communities to gradients
Pesticide toxicity of agricultural stressors and environmental variables in the region around Cluj-Napoca, Romania. Pesticide sam-
Cross-ecosystem relationships pling was done using passive samplers in the streams adjacent to spider sampling sites. To capture spiders with
Aquatic-terrestrial linkages
different traits and survival strategies, we used multiple collection methods. Community composition was best
Subsidies
explained by in-stream pesticide toxicity and shading of the stream bank, a proxy for the quality of the habitat.
Species richness and the number of spider individuals were negatively associated with in-stream pesticide tox-
icity. In contrast, mean body size and shading preference of spider communities responded strongest to shading,
whereas mean niche width (habitat preference for moisture and shading) responded strongest to the other en-
vironmental variables. Our study suggests that in-stream pesticide toxicity can influence riparian communities.
The identification of mechanisms requires further studies targeting the potential contributions of direct toxicity
and indirect effects from reduced aquatic and terrestrial prey availability.
© 2019 Elsevier B.V. All rights reserved.

⁎ Corresponding author.
E-mail address: graf-nadin@uni-landau.de (N. Graf).

https://doi.org/10.1016/j.scitotenv.2018.12.370
0048-9697/© 2019 Elsevier B.V. All rights reserved.
 N. Graf et al. / Science of the Total Environment 660 (2019) 126–135
1. Introduction
Biodiversity is declining in most biomes, with freshwater ecosys-
tems representing a system where this decline is particularly severe
(Dudgeon et al., 2006; Grooten et al., 2012; Vörösmarty et al., 2010).
In the case of running waters, these ecosystems integrate many anthro-
pogenic stressors from their upstream catchment. The major stressors
include the excessive input of nutrients, hydromorphological degrada-
tion and pollution caused by chemical inputs from point and non-
point sources. An excessive input of nutrients into freshwater systems
can result in a reduction of habitat suitability for many freshwater or-
ganisms (Smith et al., 1999). Pollutants (Baxter et al., 2005; Kraus
et al., 2017; Paetzold et al., 2011), such as pesticides (Hallmann et al.,
2014; Köhler and Triebskorn, 2013; Pekár, 2012) can cause lethal and
sub-lethal impacts on flora and fauna. Studies showed that almost half
of European water bodies are at risk from organic toxicants with pesti-
cides playing a major role (Malaj et al., 2014). The impacts of the differ-
ent stressors can be direct (i.e. direct action of a stressor on or within an
organism) or indirect (i.e. propagation of a direct effect on another or-
ganism that is connected through a biotic interaction such as competi-
tion, predation or facilitation).
Freshwater ecosystems are closely coupled with their riparian
and upstream catchment area. Organic matter from these areas en-
ters the freshwater system and constitutes an important allochtho-
nous energy source therein (Baxter et al., 2005). Conversely,
emerging insects are prey for predators in the riparian zone,
enriching the terrestrial ecosystem with energy and essential nutri-
ents (Dreyer et al., 2016; Graf et al., 2017; Kato et al., 2004;
Paetzold et al., 2005; Paetzold and Tockner, 2005; Schindler and
Smits, 2017). Spiders occur widely in riparian areas (Henschel
et al., 2001) and are important elements in terrestrial food webs,
on the one hand as predators, on the other hand as prey resource
for e.g. birds (Poulin et al., 2010). They are adapted to almost any ter-
restrial habitat and vary in their degree of habitat specialization
(Entling et al., 2011; Roberts, 1996). Some spiders build orb webs
on debris and vegetation next to the water to capture flying aquatic
emergent insects (Roberts, 1996). Free-living terrestrial spiders ob-
tain resources from aquatic prey too, but are less dependent on
aquatic subsidies than web-weaving spiders (Briers et al., 2005;
Collier et al., 2002; Sanzone et al., 2003). Studies showed that aquatic
prey consumption increases when more aquatic prey is available
(Kato et al., 2004; Kraus et al., 2014). Increasing aquatic prey avail-
ability can lead to higher spider density in riparian areas (Kraus
et al., 2014; Henschel et al., 2001; Kato et al., 2004). Conversely, a re-
duction of aquatic prey by stressors can lead to a reduction of ripar-
ian spiders. For example, contaminants reduced aquatic insects and
riparian spiders (Kraus et al., 2014; Paetzold et al., 2011). The taxo-
nomic and trait composition of spider communities can be shaped
in response to stressors, such as those related to agricultural land
use (2005; Major et al., 2006; Schmidt et al., 2008). Those traits
responding to a stressor (Violle et al., 2007), can be used to establish
trait-stressor relationships. The advantage of trait-based approaches
is that such relationships may be transferable to regions with a dif-
ferent taxonomic composition and allow for a mechanistic interpre-
tation of compositional change as well as to establishing a priori
hypotheses of relationships (Keddy, 1992; Le Viol et al., 2008;
Lambeets et al., 2008; Pekár, 2012; Schirmel et al., 2012).
Spiders can be exposed to pesticides through multiple path ways
with indirect and direct lethal and sub-lethal effects that may result in
an altered species and trait composition of spider communities
(Pekár, 2012). Most studies focused on the effects of pesticides either
on aquatic (Schäfer et al., 2012) or on terrestrial (Gibbs et al., 2009) hab-
itats in the agricultural landscape. Studies on the response of riparian
spiders to agricultural land use (Carlson et al., 2016; Krell et al., 2015;
Raitif et al., 2018) have already been conducted, but studies are lacking
that scrutinize potential field effects of pesticides on riparian organisms
127
and their trait composition. Moreover, riparian spiders may be im-
pacted by the availability of aquatic and terrestrial prey (Pekár, 2012).
Pesticides can change the community composition of invertebrates in
streams including the emergent insect composition (Liess and Von
Der Ohe, 2005) as well as terrestrial invertebrates (Geiger et al.,
2010). Thus, in-stream impacts on emerging insects may propagate to
terrestrial food webs, in particular to consumers like spiders (Pekár,
2012). However, cross-ecosystem studies on the potential propagation
of effects of pesticides in aquatic systems on riparian organisms are
lacking.
We conducted a field study on the potential response of riparian
spider communities to pesticide toxicity and other environmental
variables in Central Romania. Central Romania was selected because
remnants of traditional, low-intensity agriculture persist adjacent to
areas with high-intensity agriculture (Fischer et al., 2012), poten-
tially allowing to capture a wide gradient of pesticide toxicity. We fo-
cused on potential cross-ecosystem effects and on in-stream toxicity
assuming that effects propagate from the freshwater to the terres-
trial ecosystem.
Our research question was whether the taxonomic composition of
riparian spiders, or their number, species richness and traits (e.g. body
size, mobility), is associated with in-stream pesticide toxicity or other
environmental variables. In the absence of similar studies and given
that field studies only allow to establish associations, our study was
largely exploratory. We sampled spiders, stream pesticide concentra-
tions and monitored a range of environmental variables in Central
Romania.
2. Material & methods
2.1. Study area and sample site selection
The study area is located in Central Romania around Cluj-Napoca
in the region of Transylvania. The landscape was characterized by
Fischer et al. (2012) as a mosaic of arable fields (15% cover, less in-
tense agriculture, low use of agrochemicals expected), pastures
(40% cover, low-intensity grazing and mowing), deciduous forests
(30% cover), settlements and other minor land uses (15% cover)
(Fischer et al., 2012). We selected 19 suitable sites in the area adja-
cent to small streams (3rd to 4th Strahler order) and agricultural
fields (Fig. 1). The sites were selected to cover a gradient in agricul-
tural intensity, where land use ranged from intensive to extensive
(e.g. pasture) agriculture. The sites were selected to avoid upstream
inputs from large urban areas, treated or untreated waste water, in-
dustrial facilities and mines. Moreover, we only selected sites where
the riparian areas were suitable for sampling of the terrestrial com-
munity. The fields included in the study comprised agricultural
fields that were expected to represent high-pesticide use (e.g.
large field sizes, mechanised agriculture) and agricultural fields
that were expected to represent low-pesticide use (e.g. fields
subdivided into many small parcels, agriculture based on human
labour and working animals). Moreover, the land use ranged from
predominantly agriculture to meadows. Due to the remnants of
traditional, low-intensity agriculture in Central Romania (Fischer
et al., 2012), the site selection was expected to provide gradients
in habitat integrity, physico-chemical parameters and pesticide
exposure.
2.2. Sampling of riparian spiders
Sampling took place in May/June 2016. The spring/early summer
season was selected as this is likely the main time of pesticide applica-
tion (Szöcs et al., 2017) and the period of adult spiders (Foelix, 2011).
At each site, transects of 20 m along the streams were defined for bio-
logical sampling. To capture spiders with different traits and different
survival strategies we used multiple collection methods. We used bug
128 N. Graf et al. / Science of the Total Environment 660 (2019) 126–135
Fig. 1. Sampling sites in Romania, around Cluj-Napoca.
sieves (Sutherland, 2006), suction sampling (Sanders and Entling,
2011) and pitfall traps. Regarding sampling with bug sieves, the leaf lit-
ter of an area of 0.25 m2 was sieved (mesh of 6 mm) at six positions in
the beginning of May. During suction sampling in the end of May, the
vegetation and ground in the transect was vacuumed 50 times for 5 s
each with a modified leaf blower (STIHL SH86; Stihl). Finally, five pitfall
traps were placed with a distance of 5 m to each other and 1 m to the
streams, two on one side of the stream, three on the other side. The
traps consisted of plastic cups (volume: 0.4 L, diameter: 9.5 cm and
height: 12 cm) were filled with a 50% mixture of ethylene glycol and
water plus some detergent. Traps were deployed for seven to ten
days. Due to heavy rain fall during the first sampling period in the end
of April, the samples from pitfall traps at two sites were lost, resulting
in those two sites being excluded from the study. Furthermore, in two
and one sites only 4 and 3 traps could be recovered, respectively. The
second pitfall trap sampling in the end of May failed completely due
to flooding. We did not standardize samples to a specific time period
or trap number, because in our study the species richness and the num-
ber of spider individuals caught with the pitfall traps did not increase
statistically with trap number or time in the field given the relatively
minor (maximum of 3 days) differences between time periods (Chao
et al., 2014). Spiders of all sampling methods were combined per site,
to obtain a complete picture of the diverse living riparian spider com-
munity. Adult spiders were identified to species level and used in statis-
tical analyses, because most traits are species-specific and juveniles and
sub-adults can often not be identified to species level unequivocally
(Nentwig et al., 2017; Roberts, 1996).
2.3. Pesticide sampling and monitoring of environmental parameters
We recorded the hydromorphology of the streams and various
physico-chemical variables (see Table 2). The amount of shaded area
of the stream and riparian area was estimanted by visual observation
at noon. The land use within 200 m distance to the stream and 50 m
along both sides of the stream plus the type and amount of riparian
morphological structures within a 20 m transect were recorded. The
distances from the stream to adjacent agricultural fields on both sides
were weighted, since often these distances differed or fields were only
present on one side of the streams. They were recorded and aggregated
into a single field distance impact (fdi) factor for each sampling site to
decrease the number of explanatory variables. We calculated the dis-
tance factor as follows:
 xL   xR 
1− þ 1−
fdi ¼ 100 100 ; with x ∨x ¼ 99 if x ∨x N99 ð1Þ
L R L R
2
where xL and xR is the perpendicular distance from the stream to the ag-
ricultural field on the left and right side, respectively. The smaller the
distance from stream to field, the higher we weighted the potential im-
pact of the agricultural field on the stream, with 1 as the maximum
value, when fields directly flanked both sides of a stream (and a value
of 0.01 when fields at both sides of the stream were 100 m or more
away). The rationale for the cutoff at 100 m was that in a study a stretch
of 100 m often had the highest correlation with biological responses, or
correlated similar to wider riparian stretches (Feld, 2012). Thus, we
regarded the distance up to 100 m as the primary zone of influence in
our study.).
To determine land use in the upstream catchment of a sampling site,
we derived the stream network from a digital elevation model (DEM)
(ASTER GDEM, NASA, and METI, 2009), using the software algorithm
ATRIC (Bhowmik et al., 2015) and delineated the upstream catchment.
Subsequently, the delineated catchment polygons were intersected
with a CORINE land cover vector layer (EEA, 2007) and we calculated
the percentage per land use category (variables called catchment land
use 1 and 2, see Table 1 for further details).
Pesticide sampling was done using passive sampling in the streams
adjacent to each sampling site with polydimethylsiloxane sheets
(PDSM) (2 times for 4 weeks, for hydrophobic compounds), and
styrene-divinylbenzene (SDB) disks (4 times during heavy rain events
for 5 days; for hydrophilic compounds) (Fernández et al., 2014).
Briefly, the PDMS sheets were sampled twice for four weeks and ex-
tracted using an Accelerated Solvent Extractor (ASE, Dionex 350) over
5 cycles with methanol, cleaned up using silica gel 60 and C18 isolute
and analysed for 17 pesticides using a CG-APCI-MS/MS. We detected 4
pesticides, mainly pyrethroids. SDB disks were deployed shortly before
 N. Graf et al. / Science of the Total Environment 660 (2019) 126–135
Table 1
Traits of spiders used in the analysis with sources of trait information.
Trait Explanation Source
Body size Average female body length [mm] (Entling et al., 2010;
Nentwig et al., 2017)
Ballooning Silk-based dispersal through the air known (Bell et al., 2005; Le
or not known for the species (ballooner, Viol et al., 2008)
non-ballooner)
Shading Niche position on an open to shaded (Entling et al., 2007)
preference habitat gradient
Moisture Niche position on a moist to dry habitat (Entling et al., 2007)
preference gradient
Niche width Combined niche width towards habitat (Entling et al., 2011)
moisture and shading
Hunting Use of web for hunting prey (web builder, Cardoso et al., 2011
strategy no web builder)
heavy rainfall events (more than 10 mm/day) for five days. The disks
were extracted using acetone and methanol and measured using a
QExactive Plus Orbitrap system (Thermo Fisher Scientific Corporation)
producing HRMS and MS/MS data. We evaluated the concentrations of
187 pesticides of which 51 were detected. Details on pesticide sampling
and concentrations will be the subject to a separate publication, here we
only focus on summary information and pesticide toxicity to freshwater
invertebrates (see supplementary material). Pesticide toxicity was eval-
uated with respect to the most sensitive freshwater invertebrate spe-
cies, for which toxicity data was available (see below). We calculated
the toxicity for freshwater invertebrates because our focus was on the
potential disruption of the link between freshwater invertebrates and
riparian spiders, through potential effects on invertebrates. Moreover,
the scarcity of toxicity data for spiders prohibited the calculation of a re-
lated toxic unit. Nevertheless, the water and riparian pesticide pollution
may be associated and, therefore, we cannot exclude the possibility that
a potential relationship between pesticide toxicity and spiders repre-
sents a direct effect. The toxicity of the detected pesticides was assessed
using the logarithmic sum of the toxic unit (for details see Schäfer et al.,
2013) based on the EC50 values of the most sensitive freshwater inver-
tebrate species (Lewis et al., 2016; Malaj et al., 2014; U.S. EPA, 2018).
For the four sampling dates, we calculated the maximum of the log
sum of the toxic unit as a proxy for the maximum pesticide pollution
in the sampling period, which may directly or indirectly influence ripar-
ian spiders.
2.4. Traits
We selected one morphological (body size) and four ecological (bal-
looning, habitat niche width, shading and moisture preference, web
builder) traits, see Table 1 for details. Body size was selected because
this trait responds to environmental conditions (Entling et al., 2010;
Rico and Van den Brink, 2015; Schirmel et al., 2012). A larger body
size provides stronger protection against dehydration (Entling et al.,
2010; Schirmel et al., 2012) and pesticide contamination via e.g. spray
drift through a stronger cuticle and a higher surface-to-volume ratio
(Pekár, 2012). Larger bodied spiders might also be affected due to
slower recovery from extreme perturbations (Fox and Czesak, 2000).
Ballooning represents a dispersal trait (Bell et al., 2005) and can respond
to habitat disturbances (Entling et al., 2011), competition (Blandenier
et al., 2013; Marc et al., 1999) and prey shortage (Mestre and Bonte,
2012). Spiders might respond to stressors by emigration through bal-
looning, reducing the fraction of ballooning spiders. On the other
hand, ballooning may allow for a faster recolonization after a pesticide
impact (Bell et al., 2005; Entling et al., 2011), consequently increasing
the fraction of ballooning spiders in stressed habitats. Moisture and
shading are the most important environmental variables of Central
European spiders, and numerical values are available to describe both
the preference of spiders along these two niche dimensions (=niche
129
position; Entling et al., 2011) and their degree of habitat specialization
(=niche width; Entling et al., 2011). Habitat disturbance is expected
to favour generalist spiders with a wide niche (Entling et al., 2011). Ad-
ditionally, we included the trait of hunting strategy with the trait mo-
dalities web and non-web builder (Cardoso et al., 2011). Spiders that
build webs and are specialized to catch aquatic insects might be affected
by changes in aquatic insect emergence due to stream contaminants. On
the other hand,free hunting spiders like Pirata sp. that cross the water
surface regularly might also be affected and the related trait show a
response.
2.5. Data analysis
Pesticide toxicity in the stream and shading of the stream bank were
included as individual variables in data analysis, given our focus on the
potential relationships of pesticides in this study and since shading is
known to shape spider communities (Entling et al., 2007) as well as de-
termining the stream condition (Maloney and Weller, 2011; Naiman
and Décamps, 1997). The intensity of the agricultural land use in the
area surrounding each sampling site was not measured directly, but is
related to several measured variables such as land use type within ap-
proximately 200 m of a sampling site, percentage of shading elements
and vegetation cover within 5 m at the riparian area that were included
in the analysis (see Table 2). Overfitting models with too many vari-
ables, i.e. low ratio of sample size (i.e. number of sites) to the number
of variables as in our case, can result in spurious correlations and arte-
facts (Legendre and Legendre, 2012). We therefore limited the number
of variables entering the analysis by using dimension reduction to com-
bine multiple variables into single gradients. This was done using a
sparse principal component analysis (SPCA) in R using the package
pcaPP (Filzmoser et al., 2018). This technique improves interpretability
compared to a normal PCA by reducing the number of variables that
contribute to a gradient at the (typically minor) cost of capturing less
of the total variance of all variables (Table 2) (Zou et al., 2006). The
scores of each sampling site on the two sparse principal component
axis' were used in the subsequent regression analysis (i.e. called
(sparse) principal component regression). We note that variables omit-
ted from analysis and the two component axes still could influence our
response variables. However, field studies can only establish associa-
tions and are typically limited in sample size, prohibiting the inclusion
of all variables in statistical models. Only adult spiders were used in
data analysis.
We used Redundancy Analysis (RDA) (Oksanen et al., 2018: R pack-
age vegan) with Hellinger-transformed species data (Legendre and
Gallagher, 2001) and the four explanatory variables (two SPCA axes,
toxicity and shading) to identify the variables with the highest explan-
atory power for the composition of spider communities. Additionally, a
partial RDA (partialling out variables other than toxicity) was con-
ducted to identify the tolerant and sensitive species to toxicity
(Borcard et al., 2011). Trait data were aggregated as community-
weighted mean (CWM) per site (Laliberté et al., 2014). To identify var-
iables that are associated with total number of spider individuals, spe-
cies richness and CWM, a step wise backward model selection using
the Akaike Information Criterion or the deviance as model goodness of
fit measures was conducted. For number of adults and species richness,
we assumed a poisson distribution of the response variables and se-
lected a log link function. Given overdispersion of the model for the
number of individuals, we employed a Quasi-Poisson generalized linear
model (GLM). For the traits body size, ballooning and niche width we
assumed a Gaussian distribution and used a linear model. We also re-
port the full models as the effect sizes and standard errors after stepwise
model selection are typically overestimated and underestimated, re-
spectively. The statistical software R (Version 3.5.0 (R Development
Core Team, 2011) with the package ggplot2 were used for statistical
analyses and graphics (Wickham, 2009). We provide all code and data
under https://github.com/rbslandau/graf_spidertraits.
130 N. Graf et al. / Science of the Total Environment 660 (2019) 126–135

Table 2
Environmental variables used in data analysis with units and explanation.

Variable Unit and explanation Min – max Loading on

value SPCA axes

Shading [%] shading elementsc 0–95 Excluded

from SPCA
Toxicity Maximum of log sum toxic unit for the most sensitive freshwater invertebrate −1.591 - Excluded
0.047 from SPCA
a
Nitrite [mg/L] 0.0–0.05 No loading b
Sulfatea [mg/L] 10–200 No loading b
Catchment land use [%] Complex cultivation patterns (heterogeneous agricultural areas), land principally occupied by agriculture, with 25–51 No loading b
1c significant areas of natural vegetation and pastures in the catchment area d
Catchment land use [%] of non-irrigated arable land, vineyards, fruit trees and berry plantations in catchment area d 7.8–62 No loading b
2c
Chloridea [mg/L] 0.5–45 First axis;
0.369
a
Ammonium [mg/L] 0.05–0.40 First axis;
−0.190
Riparian cover [%] covered with shrubs at riparian area within 5 m to stream in a 20 m transect along the stream 0–58 First axis;
shrubs −0.286
Distance to field Weighted distance of streams to agriculturally used fields at right and left side of the stream, perpendicular at sampling site, 0.21–0.97 First axis;
low numbers indicate high distance 0.361
Corridor land use [%] meadows within an approximately 200 m distance to the stream in a 20 m transect along the stream 7.5–100 First axis;
meadows −0.544
Corridor land use [%] agricultural land use within an approximately 200 m distance to stream in a 20 m transect along the stream 0–90 First axis;
agriculture 0.566
Nitratea [mg/L] 0.5–1.9 Second axis;
0.410
Phosphatea [mg/L] 0.1–0.8 Second axis;
0.307
c
Riparian cover [%] covered with meadows at riparian area within 5 m to stream 0–75 Second axis;
meadows 0.700
Total area [km2] Size of catchment area 7.6–176 Second axis;
−0.501
a
Measured with compact-photometer PF-12, Machery-Nagel.
b
No loading on first two SPCA axes.
c
Catchment land use 1 consists of the CORINE codes: pastures 231, complex cultivation patterns 242, land occupied by agriculture with significant areas of natural vegetation and
pastures in the catchment area 243; Catchment land use 2 consists of the CORINE codes: non irrigated arable land 211, vineyards 221, fruit trees and berry plantations 222.

3. Results
The first axis of the SPCA explained 23.2% of the total variance of the
environmental data, whereas 12.8% was explained by the second axis.
The first axis was composed by chloride and ammonium concentration
in streams, percentage of agricultural land use and of meadows within
200 m of the stream, shrub cover at the riparian area and the field dis-
tance factor fdi representing distance of agricultural fields to the
streams. The second axis represented phosphate and nitrate water con-
centration as well as shrub cover at the meadow area as well as size of
the catchment area. Nitrite and sulphate and the land use type in the
catchment were not captured by the SPCA axes and consequently omit-
ted from analysis (Table 2). The maximum of the summed toxic unit
ranged from −1.6 to 0.05.
In total 2998 spiders were collected, of which 1222 were sampled
with pitfall traps, 72 with bug sieves and 1704 with vacuuming sam-
pling. Of these, 1757 were adults and identified to species level,
resulting in a total of 117 species at all sites. The most dominant family
was wolf spiders (Lycosidae) with 54.1% of all sampled spiders, the most
abundant species within the 117 species, was Pardosa amentata with
36.3%.
3.1. Community composition
The first axis of the RDA explained 12.9% of the total variation in
community composition, whereas the second axis explained 8.5%. The
first RDA axis was statistically significant (p = 0.013), whereas the sec-
ond RDA axis was not (p = 0.60). Community composition was best ex-
plained by the toxicity (p = 0.01, F1, 16 = 1.42) and shading (p = 0.05,
F1, 16 = 1.23) (Fig. 1). According to the partial RDA (other variables
partialled out) with toxicity (first and only RDA axis explained 8.6% of
variance), the five most sensitive species (abbreviations in Fig. 1 are ex-
plained in Supplementary material) were Pardosa prativaga (Parpra),
Diplostyla concolor (Dipcon), Clubiona reclusa (Clurec), Batyphantes
approximatus (Batapp) and Dicymbium tibiale (Dictib). However, ten ad-
ditional species exhibited very similar scores to these five most sensitive
and can be considered similarly sensitive (https://github.com/
rbslandau/graf_spidertraits). The five most tolerant species were
Rugathodes instabilis (Rugins), Trochosa spinipalpis (Trospi), Brigittea
latens (Brilat), Prinerigone vagans (Privag) and Theridiosoma gemmosum
(Thegem).
3.2. Spider richness and traits
Model building for spider species richness yielded to a best-fit model
that only contained the toxicity as explanatory variable (p = 0.016, ex-
plained deviance D2 = 0.396, n = 17). Species richness decreased with
increasing toxicity in the streams (Fig. 2.A). For the number of spider in-
dividuals, the best-fit model contained toxicity (p = 0.002, D2 = 0.481,
n = 17) (Fig. 2.B). The number of individuals decreased with increasing
toxicity. The CWM body size was only explained by the proportion of
shading in the riparian area (p = 0.001, D2 = 0.367, n = 17), with
body size decreasing with increasing shading (Fig. 2.C). CWM for shad-
ing preference correlated positively with the proportion of shading (p =
0.004, D2 = 0.441, n = 17) (Fig. 2.D). The CWM for niche width corre-
lated positively with the second SPCA axis, which included increasing ri-
parian meadow cover phosphate, nitrate and decreasing catchment size
(p = 0.020, D2 = 0.311, n = 17) (Fig. 2.E). The CWM for web building
spiders correlated with the amount of shading elements. With increas-
ing shading, more web building spiders were found (p = 0.012, D2 =
0.351, n = 17) (Fig. 3.F). The model for the community preference for
 N. Graf et al. / Science of the Total Environment 660 (2019) 126–135
Fig. 2. Redundancy Analysis (RDA) of the community composition with explanatory variables. Red names give spider species, for abbreviation see Supplementary material. Red crosses
represent spider species that were not displayed because of overlap with other spiders in the figure (the more abundant species is displayed). Blue arrows represent environmental
variables: Shading = % shading elements and max_sumTU_ms = maximal toxicity in terms of log sum of TU for most sensitive freshwater macroinvertebrate. Abbreviations explained
in supplementary material.
moisture and their ballooning ability contained only the intercept, i.e.
none of the variables exhibited explanatory power.
4. Discussion
4.1. Pesticide toxicity
We conducted a study in Central Romania in a region where tradi-
tional low intensity agriculture based on working animals and human
labour prevails alongside intensive, mechanised agriculture on the re-
sponse of spiders to different environmental variables. We expected
lower or no pesticide use in the traditional low intensity agriculture,
allowing to capture a wider gradient of in-stream pesticide exposure
compared to Western Europe. This has also been suggested in the liter-
ature e.g. Fischer et al. (2012) and Kovács-Hostyánszki et al. (2016).
However, the gradient of in-stream pesticide toxicity (in terms of
maxTU, ranging from −1.6 to 0.05) was similar to that in Western
Europe or other regions globally (Schäfer et al., 2012). This level of pes-
ticide toxicity has been found to strongly alter freshwater invertebrate
communities in field studies and controlled experiments with commu-
nities (Wijngaarden et al., 2005). Thus, it is highly likely that the stream
invertebrate communities were altered along the pesticide toxicity
gradient.
4.2. Response of spider community composition to environmental variables
The statistical models for different metrics of the riparian commu-
nity composition, i.e. RDA for community data, species richness and
total number of spider individuals, identified toxicity as most important
variable, followed by shading in the riparian area. The latter finding is in
agreement with other studies that found that shading is one of the most
important environmental variables for spiders (Entling et al., 2007;
Schirmel et al., 2012). Notwithstanding, the shading gradient lacked
data between 20% and 60% shading. Thus, the relationship with the
131
community was driven by the margins of the gradient and it remains
unknown whether the relationship is overall linear.
Several previous studies found that contaminants in aquatic sys-
tems influence adjacent terrestrial areas, either directly or via the
food webs (Hallmann et al., 2014; Kraus et al., 2017; Paetzold et al.,
2011; Poulin et al., 2010; Walters et al., 2008). The dominant explan-
atory power of pesticide toxicity including the negative association
with species richness and total number of individuals suggests direct
or indirect impacts of pesticides on spiders. However, our study does
not allow to distinguish between potential direct or indirect effects
nor between the origin of such potential effects, which could be ter-
restrial (i.e. direct exposure or reduction in terrestrial prey organ-
isms) or aquatic (i.e. reduction in aquatic prey organisms). This is
because we only quantified in-stream pesticide toxicity, and other
studies showed that this can be used to establish relationships across
the aquatic-terrestrial boundary (Hallmann et al., 2014; Poulin et al.,
2010), but in-stream pesticide toxicity may also be a proxy of terres-
trial exposure, hampering a clear attribution of observed relations
with toxicity to changes in the aquatic system. Regarding direct ef-
fects, pesticides may exert lethal and sub-lethal effects in spiders
(Pekár, 2012). For example, pesticides can cause ataxia, convulsions,
intense hyperactivity, tetanic contractions and prostration, and fi-
nally paralysis. In addition, they can disturb the water balance in spi-
ders (Pekár, 2012) or reduce feeding on prey organisms (Widiarta
et al., 2001). Substances that act neurotoxic can reduce the number
of spider individuals (Desneux et al., 2007). Additionally, oral uptake
of pesticides can decrease immune capacity of spiders and can result
in lower immune capacity leading to an increase in diseases and to
mortality (Punzo, 1997), slower development and lower reproduc-
tion or higher susceptibility to predators. Toxicity information on
species that responded to the pesticide toxicity gradient in the
RDA, i.e. either being sensitive or tolerant, are lacking. However,
such data would allow to evaluate whether the effects are direct. Ad-
ditionally, toxicity data from the terrestrial area could help to better
separate direct and indirect effects of toxicity. However, beside the
132 N. Graf et al. / Science of the Total Environment 660 (2019) 126–135

Fig. 3. Relationships of spider species richness (A), number of individuals (B) and trait composition (body size: C, shading preference: D, niche width: E, web builder: F) with
environmental variables and the confidence band for each model.

remaining uncertainties regarding the underlying mechanisms of stronger control of the conditions in the field or laboratory are re-
the observed associations between toxicity and spider responses, quired to identify the underlying mechanisms.
the consistency and plausibility of the effect directions suggests Previous studies showed a positive relationship between aquatic
that this relationship is not an artefact. Further studies with a prey availability and aquatic prey consumption with spider abundance
 N. Graf et al. / Science of the Total Environment 660 (2019) 126–135
in riparian areas (Burdon and Harding, 2008; Henschel et al., 2001). A
manipulative reduction of aquatic insects demonstrated a decrease
of spiders in the riparian area (Kato et al., 2004; Marczak and
Richardson, 2007). Regarding indirect effects, pesticides can reduce
food for the prey of spiders (Lensing et al., 2005) and the spider prey it-
self, both processes potentially reducing prey availability (Pekár, 2012).
Discharge of mining and a gradient of metal contaminants lead to a re-
duction of aquatic insects and spiders (Kraus et al., 2014; Paetzold et al.,
2011). Comparable results were found for other organisms depending
on aquatic systems. For instance, in-stream concentrations of pesticides
such as neonicotinoids were associated with a decline in insectivorous
birds, caused by a decline in insect macrofauna (Hallmann et al., 2014).
4.3. Response of spider traits to environmental variables
The community-weighted mean body size correlated negatively
with shading of the stream bank. Body size is a trait that responds to
micro climate (Entling et al., 2010; Schirmel et al., 2012). Dry and hot
conditions are preferred by larger spider species, due to their compact
and waterproof cuticle and their lower surface area-to-volume ratio,
which provides higher desiccation resistance (Remmert, 1981; Entling
et al., 2010; Schirmel et al., 2012).
Niche width correlated positively with the second SPCA Axis. The
second axis represented high phosphate and nitrate water concentra-
tion, shrub cover at the meadow area as well as a low catchment area.
Meadows are species rich habitats and nutrients can increase the abun-
dance of prey organisms. Species with wider niches have higher dis-
persal (balloooning) propensities (Entling et al., 2011). Even though
we could not find a correlation between CWM for ballooning and ex-
planatory variables, the communities' ballooning ability and the niche
width correlated positively (p = 0.03, t = 2.44, n = 17, corr = 0.533).
Consequently, these correlations might lead to an increase of ballooning
spiders and spiders with wide niche preferences, respectively, in prey
rich areas.
The CWM for shading was positively associated with the shading of
the stream bank. This relationship could be regarded as trivial, but in-
creases the confidence regarding the interpretation of the other rela-
tionships. None of the explanatory variables were selected in the
statistical model for the CWM moisture preference. As all study sites
were placed next to streams, moisture preference may not respond to
the captured environmental gradients because riparian habitats tend
to be moister than other habitats.
The number of CWM for spiders hunting with webs increased with
the amount of shading elements. In total the number weighted mean
for web hunting spiders in generally very low and only at one site pos-
itive. Shading is a representative variable for attachment options of
webs for spiders. It comes with no surprise that with additional attach-
ment options for web weaving spiders, they increase whereas they de-
crease with decreasing ratios of shrubs, trees and bushes.
We did not find a relationship between traits and pesticide toxicity.
This may be because the chosen traits are not suitable to establish rela-
tionships with toxicity. For example, a decrease of species richness and
the number of individuals by pesticides (irrespective whether indirect
or direct), may free niche space that can be occupied by species more
tolerant to pesticides, potentially exhibiting a similar trait profile as
the traits under scrutiny are not selected by toxicity.
5. Conclusion
Our study is one of the first to address the relationship between pes-
ticide toxicity and riparian spider biodiversity. Our research question fo-
cused on relationships between taxonomic composition of riparian
spiders, their number, species richness and traits, and in-stream pesti-
cide toxicity and other environmental variables. Pesticide toxicity had
the highest association with the taxonomic composition of riparian spi-
ders and their number and species richness. In contrast, the available
133
traits were mostly associated with variables such as habitat structure,
land use and micro climate. More controlled field and laboratory studies
on the relationships between pesticide toxicity and spiders in riparian
areas are needed to differentiate between direct and indirect effects
and to clarify underlying mechanisms, which may be important to pre-
dict the future development of spider communities in face of the world-
wide increase in the use of agricultural pesticides (Bernhardt et al.,
2017).
Acknowledgment
Three anonymous referees provided valuable suggestions that im-
proved the manuscript. The authors thank the Department of Environ-
mental Sciences of the Swiss Federal Institute of Aquatic Science and
Technology (Eawag) namely Bernadette Vogler, Birgit Beck, Heinz
Singer and Juliane Hollender for support with pesticide analysis under
their supervision. We thank Jörn Buse for help with the site selection
and general study design, Sophia Schmidt and Sebastian Marx for assis-
tance as well as Lucas Streib for map creation. This work has been sup-
ported by the Ministerium für Wissenschaft, Weiterbildung und Kultur
Rheinland-Pfalz, Germany, (“Research initiative, project AufLand”) and
by the German Research Foundation (DFG: SCHA 1720/12-1).
Authors' contributions
NG, MHE and RBS designed the study. NG, RBS, KPB and MC selected
the sites. KPB and MCassisted with logistics. NG sampled invertebrates,
conducted the statistical analysis and drafted the manuscript. PD and
NG prepared samples and identified the spiders. VCS conducted the pes-
ticide analysis and created the pesticide gradient. VCS, and AS provided
data from toxicity tests. ML, MHE, ES and RBS provided feedback on the
statistical analysis. All authors revised the manuscript and gave final ap-
proval for publication and agree to be accountable for all aspects of the
work in ensuring that questions related to the accuracy or integrity of
any part of the work are appropriately investigated and resolved.
Appendix A. Supplementary data
Supplementary material like supporting information, raw data and R
code used for this study can be found at https://github.com/rbslandau/
graf_spidertraits.
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