Invasive Species Mediate Insecticide Effects on Community and

Subscriber access provided by Queen Mary, University of London

Ecotoxicology and Human Environmental Health

Invasive species mediate insecticide effects on community and ecosystem functioning. Andreia CM Rodrigues, Ana L Machado, Maria D Bordalo, Liliana Saro, Fátima C. P. Simão, Rui MJ Rocha, Oksana Golovko, Vladimir Zlabek, Carlos Barata, Amadeu M.V.M. Soares, and João L. T. Pestana Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b00193 • Publication Date (Web): 22 Mar 2018 Downloaded from http://pubs.acs.org on March 24, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 37

Environmental Science & Technology

1

Abstract Art:

2

3

1 ACS Paragon Plus Environment


4

Title: Invasive species mediate insecticide effects on community and ecosystem functioning.

5

Andreia C. M. Rodrigues1,3*, Ana L. Machado1, Maria D. Bordalo1, Liliana Saro1, Fátima C. P.

6

Simão1, Rui J. M. Rocha1, Oksana Golovko2, Vladimír Žlábek2, Carlos Barata3, Amadeu M. V. M.

7

Soares1, João L. T. Pestana1

8

1

9

Santiago, 3810-193 Aveiro, Portugal

Page 2 of 37

Departamento de Biologia & CESAM, Universidade de Aveiro, Campus Universitário de

10

2

11

South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Vodnany,

12

Czech Republic

13

3

14

Spain

15

*Correspondence and requests for materials should be addressed to A.C.M.R. (email:

16

[email protected])

University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters,

Department of Environmental Chemistry (IDAEA-CSIC), Jordi Girona, 18-26, 08034 Barcelona,

17


Page 3 of 37


18

ABSTRACT:

19

Anthropogenic activities increase pesticide contamination and biological invasions in freshwater

20

ecosystems. Understanding their combined effects on community structure and on ecosystem

21

functioning presents challenges for an improved ecological risk assessment. This study focuses on

22

an artificial stream mesocosms experiment testing for direct and indirect effects of insecticide

23

(chlorantraniliprole – CAP) exposure on the structure of a benthic macroinvertebrate freshwater

24

community and on ecosystem functioning (leaf decomposition, primary production). To understand

25

how predator identity and resource quality alter the community responses to chemical stress, the

26

mediating effects of an invasive predator species (crayfish Procambarus clarkii) and detritus

27

quality (tested by using leaves of the invasive Eucalyptus globulus) on insecticide toxicity were

28

also investigated. Low concentrations of CAP reduced the abundance of shredders and grazers,

29

decreasing leaf decomposition and increasing primary production. Replacement of autochthonous

30

predators and leaf litter by invasive species decreased macroinvertebrate survival, reduced leaf

31

decomposition and enhanced primary production. Structural equation modeling (SEM) highlighted

32

that CAP toxicity to macroinvertebrates was mediated by the presence of crayfish or eucalypt leaf

33

litter which are now common in many Mediterranean freshwaters. In summary, our results

34

demonstrate that the presence of these two invasive species alter the effects of insecticide exposure

35

on benthic freshwater communities. The approach used here also allowed to mechanistically

36

evaluate the indirect effects on ecosystem functional endpoints of these stressors and of their

37

interaction, emphasizing the value of incorporating biotic stressors in ecotoxicological experiments.



Page 4 of 37

38

INTRODUCTION:

39

Freshwater ecosystems provide essential resources for humans and are considered rich habitats

40

with a great diversity of organisms. Nevertheless, these ecosystems are frequently exposed to

41

several anthropogenic pressures that lead to their degradation and loss of biodiversity, making their

42

recovery one of the priority actions of governments and management policies nowadays 1,2. A great

43

challenge for risk assessment is set by the combinations of pesticides and natural stressors that are

44

probable to co-occur and whose combined effects on freshwater communities are difficult to

45

predict 3–5. Hence, there is a growing awareness that pesticide effects may be misjudged due to

46

interactions with biotic stressors and, increasing the complexity of ecotoxicology testing is

47

essential for a better ecological risk assessment 3,6,7. Moreover, biological invasions are nowadays

48

of the most significant causes of biodiversity loss and ecosystem alterations within freshwaters 8,

49

which will further add complexity to the assessment of contaminants effects. Because shifts in

50

species composition have been shown to affect community dynamics 9 and ecosystem functions,

51

such as litter decomposition and productivity 10, and thus it is critical to investigate the potential

52

role of invasive species in altering the community context as well as becoming an important

53

mediator of contaminant effects on natural ecosystems 4,11.

54

In freshwater ecosystems and within detritus based food chains, density and trait mediated effects

55

of predation and resource quality (leaf litter) may shape ecosystem functioning 12,13. These natural

56

biotic stressors and the effects of non-indigenous predators and leaf litters are thus potential

57

mediators of changes on organisms and populations susceptibility to chemicals 14–17.

58

Predators of invertebrate detritivores are known to influence trophic dynamics and elemental

59

cycling through effects on leaf litter decomposition, a key ecosystem process in many freshwater

60

systems 16. However, the mechanisms of such influences are not well understood and there is an

61

increased debate about the strength of non-trophic interactions and habitat complexity 18. Among

62

these predators, omnivorous macroinvertebrates are usually top consumers that may decouple

63

cascading trophic chains through both direct effects on the food resource and indirect effects on


Page 5 of 37


64

intermediate consumers 19,20. For this study we selected the red swamp crayfish, Procambarus

65

clarkii, native to northeastern Mexico and southcentral USA that has become a dominant predator

66

in several European rivers 21. This crayfish has many properties of a successful invader (plastic life

67

cycle, generalist and opportunistic feeding habits, behavioral plasticity when coping with other

68

predator species), allowing rapid dispersion and high tolerance towards environmental extremes

69

22,23

70

Regarding leaf litter, the replacement of native riparian vegetation by exotic plantations that is

71

occurring worldwide poses an extra challenge to freshwater benthic detritivore communities,

72

especially in low order streams 24,25. The input of allochthonous leaf litter of different nutritional

73

quality, litter fall timing and quantity has the potential to alter benthic invertebrate assemblages and

74

the structure as well as functioning of these communities 16,26. Eucalyptus globulus, which is widely

75

planted in Portugal, has demonstrated invasive behaviour, since it can grow aggressively outside

76

plantations 27,28. Nowadays, E. globulus is frequently found on the riparian vegetation throughout

77

southern European countries, replacing native species such as Alnus glutinosa and changing

78

organic matter dynamics 29,30. Eucalyptus leaves have lower nutritional quality for freshwater

79

detritivores and higher quantities of essential oils as well as polyphenols (that are toxic to most

80

freshwater organisms), than leaves of European native species (e.g., A. glutinosa) 31–33..

81

The present study intends to evaluate the combined effects of insecticides and invasive species on

82

freshwater benthic macroinvertebrate community structure and function, with special focus on

83

detritus-based stream food webs 34. Chlorantraniliprole (CAP), which belongs to the anthranilic

84

diamides pesticides group, was used as model insecticide. CAP readily won the pesticides market

85

due to successful action against target species at low doses and high selectivity toward ryanodine

86

receptor of insects 35,36. Nevertheless, feeding and development impairment have already been

87

reported for freshwater non-target invertebrates exposed to environmentally relevant CAP

88

concentrations 35,37–40.

.



Page 6 of 37

89

To test the hypothesis that direct and indirect effects of environmentally relevant insecticide

90

exposure on macroinvertebrates communities and on ecosystem functioning, can be mediated by

91

predator identity and/or resource quality, we performed mesocosms experiments where these

92

different stressors were combined. For that, we exposed a natural benthic invertebrate community

93

to different combinations of stressors: insecticide exposure (0 vs 2 µg/L CAP), predator identity

94

(native odonate Cordulegaster boltonii vs invasive crayfish P. clarkii) and different leaf litter

95

(leaves from the native A. glutinosa vs the invasive E. globulus). Direct and indirect effects of

96

insecticide exposure and the presence of invasive species as well as of their interactions, were

97

assessed on community structure (detritivore abundances) and on ecosystem functional endpoints

98

(leaf decomposition and primary production). We predicted that pesticide exposure, replacement of

99

dragonfly predator by crayfish and of alder leaf litter by eucalypt leaf litter, and their combination

100

would negatively affect leaf decomposition while enhancing primary production either though

101

density or trait mediated effects in invertebrate community. Primary producers and predator species

102

are, in general, more tolerant to this insecticide 35 so, no direct effects of CAP were expected for

103

these groups.

104

Structural Equation Modeling (SEM) was used to mechanistically discriminate and relate indirect

105

effects and interactions. Through path analysis we can quantify the relative weight of direct,

106

indirect and interaction effects of the studied stressors on community structure, linking changes in

107

different invertebrate functional groups to alterations on ecosystem functional parameters.

108

METHODS

109

Macroinvertebrate and leaf collection.

110

The Mau river (Sever do Vouga, 40º 44’ 21.2’’ N, 8º 24’ 6.9’’ W, Portugal) was selected for

111

invertebrate sampling as this stream presents a good ecological quality status 41. Prior to the

112

experiment and to determine the composition of benthic macroinvertebrate communities in the

113

chosen aquatic system, four quantitative samples were taken using an enclosed surber sampler


Page 7 of 37


114

(0.09 m2, 500 µm mesh), in winter (January 2016), from slow-flow riffle habitats.

115

Macroinvertebrate samples were preserved in 70% ethanol and were identified to family level,

116

using a stereomicroscope. The density of each taxon in the natural habitat was then calculated,

117

based on the mean values of the four samples. These density values were then used as reference to

118

distribute the different taxa among the artificial streams, according to their natural densities.

119

Juveniles of Procambarus clarkii Girard were collected using funnel traps in channels of Ria de

120

Aveiro, central Portugal. Organisms were acclimated to laboratory conditions for at least one

121

month before the experiment (15 ºC, 16 h light: 8 h dark photoperiod).

122

Alnus glutinosa (L.) Gaertn leaves were collected soon after senescence from the riparian

123

vegetation at São Pedro de Alva (40º 16’ 37’’N, 8º 11’ 51.72’’ W), central Portugal. Leaves of

124

Eucalyptus globulus Labill. were abscised during autumn at Boialvo (40º 30’ 4.2’’ N, 8º 20’ 37.0’’

125

W), central Portugal. Both types of leaves were air dried and stored in darkness.

126

Description of the mesocosm experiments.

127

The present study was conducted in an indoor modular mesocosms system, in a total of 24 glass

128

artificial streams (individual streams: 2 m length, 0.200 m width, 0.225 m depth) on recirculated

129

water regime. Flow in each artificial stream was maintained at a constant rate of 3.75 L/min,

130

similar to the hydraulic conditions present in nearby natural riffles. The aqueous media used was

131

artificial pond water (APW) 42, enriched with phosphate, nitrate and silicate to meet realistic

132

concentrations of these minerals in Mau river water 41. The mesocosm room was maintained at

133

constant temperature of 15 ± 1 ºC and with a photoperiod of 16 h light: 8 h dark, to simulate field

134

conditions. The artificial streams were filled with enriched APW medium (~ 280 L) and

135

commercial river sand (~ 2 cm layer), which was previously heated at 500 ºC for 4 h to avoid

136

contamination with possible adsorbed compounds. The varied granulometry of the commercial

137

river sand suits the different needs of the species in our community.



Page 8 of 37

138

Alder and eucalyptus leaves were previously conditioned for 14 days in separate plastic buckets

139

with aerated natural river water, which allowed leaching33 and a similar microbial conditioning 43.

140

Leaves were then dried at 50 ºC for 4 days, weighed and placed in the respective experimental

141

streams in leaf packs with a coarse mesh (10 mm mesh size). The mean initial weight of alder and

142

eucalypt leaves available per artificial stream was 1660 ± 54.4 mg and 1696.6 ± 68.3 mg (mean ±

143

SD), respectively. Ten unglazed ceramic tiles (20 cm2) were added along each artificial stream as

144

standardized substrate for posterior periphyton sampling. Stones from the Mau River were scraped

145

with toothbrushes and washed with water from the site to obtain a final volume of ~ 5 L of

146

concentrated biofilm. In the same day of collection, 100 mL of this concentrated inoculum of

147

biofilm was added to each artificial stream and recirculated through the experimental streams for

148

15 days to allow for biofilm colonization.

149

On the 15th day (day 0 of exposure period), benthic macroinvertebrates were collected by kick-

150

sampling in riffle and pool habitats using a 500 µm mesh pond net in Mau river in February 2016.

151

Macroinvertebrates were transferred in river water to the laboratory, immediately sorted by taxa

152

and placed into the artificial streams based on the previously calculated densities. Individuals of the

153

same taxa were selected by eye based on their similar size and distributed in equal numbers per

154

artificial stream. A total number of 217 macroinvertebrates were thus distributed per artificial

155

stream, representing 17 taxa, with the main representative functional feeding groups in terms of

156

abundance being shredders (62 individuals), collectors (95 individuals) and grazers (49 individuals

157

per artificial stream) (SI Table S1). The macroinvertebrate community was exposed for 7 days to

158

the combinations of: insecticide (0 or 2 µg/L CAP), predator identity (the native Cordulegaster

159

boltonii or the invasive Procambarus clarkii) and leaf litter from two different tree species (the

160

native A. glutinosa or the invasive E. globulus) (Figure 1). Each treatment combination was

161

replicated in 3 artificial streams. One odonatan or one crayfish were allocated per artificial stream,

162

with mean fresh weight of 712 ± 114 mg and 744 ± 177 mg (mean ± SD), respectively.


Page 9 of 37


163 164

Figure 1. Schematic representation of experimental design.

165

End of the experiment assessment.

166

Water physico-chemical parameters (temperature, pH, conductivity and dissolved oxygen) of the

167

artificial streams were assessed every two days and remained similar across all treatments

168

throughout the trial, with mean values of: 15.3 ± 0.2 ºC, pH of 8.0 ± 0.4, 810 ± 13.7 µS/cm, 9.7 ±

169

0.3 mg O2/L.

170

After 7 days of exposure period, leaf packs were carefully rinsed in distilled water, sediment

171

particles removed with a soft paint brush, and the attached invertebrates retrieved. Remaining leaf

172

material was dried at 50 ºC for 4 days and weighed. Ceramic plates were removed from each

173

artificial stream, scrubbed with toothbrushes and rinsed with water to individual plastic flasks.

174

Samples for periphyton identification were fixed with 5 % formaldin and stored in darkness.

175

Samples for chlorophyll measurement were kept in the dark, at 4 ºC for 24 h. These samples were

176

then filtered through GF/C filters (1.2 µm; Whatman Inc.) and stored in darkness at – 20 ºC until

177

analysis. Extraction was performed with 90 % acetone and chlorophyll a measured

178

spectrophotometrically following Jeffrey and Humphrey (1975). Chlorophyll a concentration was

179

used as an estimation of periphyton primary production. All the macroinvertebrates per artificial



Page 10 of 37

180

stream were collected by sieving the sediment and then preserved in 70 % ethanol for later

181

identification, using a stereomicroscope.

182

Insecticide contamination.

183

A stock solution of CAP (analytical standard, CAS No. 500008-45-7, Sigma-Aldrich) was prepared

184

in analytical grade acetone, protected from light to avoid degradation. Water was spiked to a

185

nominal concentration of 2 µg/L CAP in experimental streams (keeping acetone below 0.01 %).

186

The selected nominal CAP concentration represents a realistic measured concentration in

187

freshwaters 40 and also a sub-lethal concentration for most of the detritivore freshwater

188

invertebrates 35,37,38. Qi and Casida 45 found no significant binding activity of CAP to ryanodine

189

receptor of a decapoda species. Also, Barbee et al. 46 tested the sensitivity of P. clarkii to CAP and

190

reported a no-observed effect concentration of 480 µg/L. The native representative predator

191

Cordulegaster boltonii showed similar tolerance to CAP exposure: > 640 µg/L (acute exposure)

192

and > 96 µg/L (feeding inhibition test) (personal observation, data not shown). Nominal CAP

193

concentrations in overlying water (sampled at 2 h, 96 h and 7 days of exposure) were verified by

194

chemical analysis. All samples were frozen at − 20 °C and sent in dry ice for analysis. Liquid

195

chromatography mass spectrometry (LC-MS) was used to quantify CAP on water samples (detailed

196

description of the chemical analysis in SI).

197

Statistical analysis.

198

A t-test compared the number of organisms per stream on day 0 and day 7 in the control streams

199

(no-CAP and no invasive species presence) to exclude effects on survival of organisms due to

200

factors other than the tested stressors.

201

Differences in macroinvertebrate community structure among treatments (CAP exposure, predator

202

identity and leaf litter type) were first tested globally by Permutational Multivariate Analysis of

203

Variance (PERMANOVA) using the absolute abundance of all the present taxa. Bray-Curtis

204

dissimilarity was used as a distance measure and the significance of the analysis was tested using


Page 11 of 37


205

Monte Carlo permutation method with 999 permutations 47. These analyses were performed on R

206

software 3.2.2 version, from The R Foundation for Statistical Computing (Vienna, Austria), using

207

the ‘vegan’ package 48.

208

Additionally, for functional analysis, macroinvertebrates were grouped into three functional

209

feeding groups according to Usseglio‐Polatera et al.49 and Tachet et al.50: shredders

210

(Sericostomatidae, Lepidostomatidae, Calamoceratidae and Limnephilidade), collectors

211

(Ephemeridae and Chironomidae) and grazers/scrapers (Habrophlebiidae, Baetidae and

212

Heptageniidae). The abundance of individuals in each feeding group per artificial stream was

213

calculated. These functional groups include the main representative taxa of our community,

214

accounting for ~ 88 % of initial number of the organisms per stream, and also three important

215

feeding groups regarding leaf decomposition/ grazing and nutrient cycling 51.

216

Macroinvertebrates abundances per functional feeding group, leaf decomposition and periphyton

217

primary production data were analyzed by three-way analysis of variance (ANOVA). This

218

procedure identified statistically significant differences between treatments with contamination by

219

CAP, resource quality and predator identity as fixed factors. Normality of data was assessed using

220

the Shapiro-Wilk normality test and residual plots, plus homoscedasticity was verified with the

221

Brown-Forsythe test. The significance level was set at 0.05 and calculations were performed using

222

GraphPad Prism version 7.00 for Windows (GraphPad Software, La Jolla California USA).

223

A Structural Equation Modelling (SEM) model was used to investigate mechanistic pathways that

224

may explain the direct, indirect and total effects 52,53 of CAP exposure, predator identity and leaf

225

litter type on community composition (abundance of shredders, collectors and grazes), and on

226

ecosystem functions (leaf decomposition and primary production). By using SEM to interpret the

227

mesocosm results, a refined hypothesized model was obtained to evaluate the effects of insecticides

228

and invasive species at the community and ecosystem levels. Path coefficients (partial multiple

229

regression coefficients), with the values of each variable standardized to a common metric, allow to

230

compare the relative importance of each effect. All response variables were previously normalized



Page 12 of 37

231

so that their responses had equal weight in the analysis. Since we tested for effects of absence/

232

presence of invasive species and their interaction with insecticide exposure (single concentration)

233

against control treatments (autochthonous species and no insecticide exposure) all exogenous

234

variables (CAP, crayfish and eucalypt leaves) in the model were set as either 0 or 1. The same for

235

interaction variables with 1 representing the treatments were 2 stressors were simultaneously

236

present (CAP and eucalyptus, CAP and crayfish or eucalyptus and crayfish). Indirect effects of a

237

variable on another were calculated as the sum of the products of direct path coefficients 54.

238

Strength and sign of the total effect of a stressor on one variable were calculated as the sum of both

239

direct and indirect effects 54. The fit of the path model for our data was tested using four goodness-

240

of-fit measures: the χ2 test value testing the similarity between the observed and the predicted

241

covariance matrices; the root of mean square error of approximation (RMSEA) where values ≤

242

0.05 indicate a good fit; the comparative fit index (CFI) where a value ≥ 0.95 combined with the

243

standardized root-mean-squared residual (SRMR) value ≤ 0.08 is indicative of a good fit 55.

244

Preliminary SEM models testing for the interactions between functional feeding groups were

245

designed and tested, being the final model selected based on a likelihood ratio test (p < 0.05) and

246

the goodness of fit values described above. These analyses were performed with R software 3.2.2

247

version, from The R Foundation for Statistical Computing, using the ‘lavaan’ package 56.

248

RESULTS

249

Chemical fate of CAP and exposure concentrations.

250

Measured concentrations of CAP are presented in Table 1. After 7 days measured concentrations of

251

CAP were still on average 77 % of initial ones, which means that CAP was quite stable in the

252

studied mesocosms and in agreement with previous reported stability of CAP in water under lower

253

temperatures and pH of 8.0 57.

254 255


Page 13 of 37


256 257

Table 1. Chlorantraniliprole concentrations measured in overlying water (µg/L; mean ± SD) after 2

258

h, 96 h and 7 days of experimental exposure.

259 CAP treatments

Measured concentration (µg/L) 2h 96 h 7 days (n = 3) (n = 1) (n = 3)

Alder + C. boltonii

1.63 ± 0.21

1.6

1.29 ± 0.37

Alder + P. clarkii

1.40 ± 0.26

1.5

1.16 ± 0.47

Eucalypt + C. boltonii

1.65 ± 0.14

1.6

1.23 ± 0.36

Eucalypt + P. clarkii

1.50 ± 0.14

1.6

1.11 ± 0.43

260 261

Global analysis of effects on the macroinvertebrate community structure and composition.

262

No significant differences (t4 = 1.732, p = 0.158, η2 = 0.429) between invertebrates’ abundances at

263

day 0 and day 7 were observed in the streams simulating natural conditions (A. glutinosa as leaf

264

litter and predation by the native C. boltonii).

265

PERMANOVA results showed that significant alterations of macroinvertebrate community were

266

due to crayfish presence (F1,23 = 25.42, p = 0.001), eucalyptus presence (F1,23 = 3.65, p = 0.027) and

267

due to the predator - leaf - CAP interaction (F1,23 = 2.89, p = 0.049). No significant effects of CAP

268

or two-way interactions were revealed by this analysis (detailed information in SI Table S2).

269

Changes on community structure and function.

270

The mean values of abundance for the three functional groups, changes in leaf decomposition and

271

primary production (measured as chlorophyll a concentration) after 7 days of exposure are

272

presented in Figure 2a-e. The presented SEM model provided good fit to the data: χ2 = 0.82, df = 6,

273

p = 0.99; RMSEA = 0.00, GFI = 1.00 and SRMR = 0.005, with the changes on the abundances of

274

shredders, collectors and grazers, leaf decomposition and primary production being well explained



Page 14 of 37

275

by the model (R2 range: 0.561–0.974, Figure 3). In general, results of Three-way ANOVA (Table

276

2) and SEM analysis (Table 3) agree in the main direct effects of CAP, resource quality and

277

predator identity to the three functional groups of macroinvertebrates and leaf decomposition.

278

Shredders abundance decreased significantly (p < 0.05) upon exposure to CAP, by the presence of

279

eucalypt leaves and crayfish (Table 2). The SEM analysis showed that both CAP exposure and

280

eucalypt leaves contribute similarly to the observed decrease in shredders abundance with a

281

standardized path coefficient (r) = - 0.662 (Figure 3, Table 3). SEM analysis also identified a

282

negative effect caused by crayfish, i.e. decreases in shredders abundance (r = - 0.397, Figure 3,

283

Table 3).

284

The presence of crayfish and eucalypt leaf litter also induced negative direct effects on collectors (r

285

= - 1.312 and r = - 0.270, respectively) while no direct effects of CAP exposure were detected

286

either by SEM analysis nor by linear models (Figure 3, Table 2 and 3). Crayfish presence (r = -

287

1.172) and CAP exposure (r = - 0.486) also had negative effects on grazers (Figure 3 Table 2,3).

288

SEM analysis revealed that CAP direct effects on collectors were stronger in the presence of

289

eucalypt leaf litter (interaction effect, r = - 0.329, Figure 3, Table 3). On the other hand, crayfish

290

direct negative effects on grazers and collectors were reduced under CAP exposure and the

291

presence of crayfish (CAP × Crayfish, interaction effects: r = + 0.421 for both invertebrate groups,

292

Figure 3, Table 3). These negative effects of crayfish on collectors were also greatly reduced in

293

treatments with eucalyptus leaf litter (Eucalyptus x Crayfish, interaction effects r = + 0.467, Figure

294

3, Table 3).

295


Page 15 of 37


296 15 ACS Paragon Plus Environment


Page 16 of 37

297

Figure 2. Effects of chlorantraniliprole (CAP) exposure and leaf litter type (Alnus glutinosa or

298

Eucalyptus globulus) on: the abundance (no. of individuals) of macroinvertebrates per feeding

299

group (a) shredders, (b) collectors and (c) grazers; (d) leaf decomposition as leaf mass loss (mg)

300

and (e) primary production, as chlorophyll a concentration (mg/cm2), in streams mesocosms with a

301

native and invasive predator (Cordulegaster boltonii or Procambarus clarkii, left and right

302

columns, respectively), after 7 days exposure. All values are presented as mean ± SE.

303

Table 2. Three-way ANOVA results testing for effects of effects of chlorantraniliprole (CAP)

304

exposure, crayfish (C) and eucalyptus (E) presence, and their interactions on shredders, collectors

305

and grazers abundances, leaf decomposition and primary production. Factor

df

F

p

R2

CAP

1, 16

5.59

0.031

0.110

Eucalypt

1, 16

14.31

0.002

0.281

Crayfish

1, 16

5.59

0.031

0.110

C × CAP

1, 16

2.012

0.175

0.039

E × CAP

1, 16

0.894

0.358

0.018

E×C

1, 16

0.224

0.643

0.004

CAP × E × C

1, 16

6.36

0.023

0.125

Collectors

CAP

1, 16

2.521

0.132

0.021

abundance

Eucalypt

1, 16

4.28

0.055

0.036

Crayfish

1, 16

75.84

< 0.001

0.640

C × CAP

1, 16

4.28

0.055

0.036

E × CAP

1, 16

7.00

0.018

0.059

E×C

1, 16

8.613

0.010

0.073

CAP × E × C

1, 16

0.021

0.887

1.8*10-4

CAP

1, 16

0.754

0.398

0.010

Eucalypt

1, 16

0.754

0.398

0.010

Crayfish

1, 16

48.2

< 0.001

0.633

C × CAP

1, 16

1.572

0.228

0.021

Endpoint Shredders abundance

Grazers abundance


Page 17 of 37


Leaf decomposition

Primary production

E × CAP

1, 16

4.502

0.049

0.059

E×C

1, 16

1.34

0.264

0.018

CAP × E × C

1, 16

3.014

0.102

0.040

CAP

1, 16

1.186

0.192

0.003

Eucalypt

1, 16

649.2

< 0.001

0.964

Crayfish

1, 16

0.002

0.968

2.5*10-6

C × CAP

1, 16

0.002

0.967

2.6*10-6

E × CAP

1, 16

2.771

0.116

0.004

E×C

1, 16

0.236

0.634

3.5*10-4

CAP × E × C

1, 16

3.203

0.093

0.005

CAP

1, 16

0.331

0.573

0.004

Eucalypt

1, 16

9.677

0.007

0.122

Crayfish

1, 16

35.74

< 0.001

0.450

C × CAP

1, 16

0.384

0.544

0.005

E × CAP

1, 16

12.36

0.003

0.156

E×C

1, 16

2.869

0.110

0.036

CAP × E × C

1, 16

2.005

0.176

0.025

306 307

Decomposition of eucalypt leaf litter was significantly lower when compared with alder leaves (r =

308

- 0.943, Figure 3). Three-way ANOVA’s and SEM results are in agreement since non-significant

309

direct effects of CAP or predator identity were obtained (Figure 2,3, Table 2 and 3). However,

310

SEM analysis also show weak negative effects of CAP exposure on leaf decomposition (Table 3,

311

Figure 3). Negative indirect effects of the presence of crayfish through effects on shredders and

312

collectors’ abundances on leaf decomposition were also identified (r = - 0.182, Table 3).

313

ANOVA’s showed that both eucalypt leaf litter and crayfish presence induced positive effects on

314

primary production (i.e. higher chlorophyll a concentration, Table 2), with SEM analysis indicating

315

positive albeit non-significant total effects of all stressors on primary production (Table 3).

316

Moreover, SEM analysis identify eucalypt leaf litter presence as the main stressor affecting primary

317

production (Figure 3, Table 3). These positive effects of eucalypt leaf litter on primary producers



Page 18 of 37

318

were stronger when crayfish were present (Eucalyptus × Crayfish, r = + 0.632, Figure 3, Table 3)

319

but were reversed under CAP exposure (Eucalyptus × CAP, r = - 0.353, Figure 3, Table 3). A

320

complete description of direct, indirect and total effects is presented in Table 3.

321 322

Figure 3. Graphical representation of the supported SEM model for chlorantraniliprole (CAP), leaf

323

litter and predator identity and their combined effects. Standardized path coefficients and variation

324

explained by the model are given for response variables. Thickness of the arrows is proportional to

325

the strength of the standardized path coefficient. * p < 0.05, ** p < 0.001.

326 327

Table 3. Estimates for standardized path coefficients (r) of the structural equation model for direct,

328

indirect, and total effects of chlorantraniliprole (CAP) exposure, crayfish (C) and eucalyptus (E) presence,

329

and their interactions on macroinvertebrate abundance, leaf decomposition and primary production. (SE:

330

standard error) Significant (p < 0.05) values are stressed in bold.


Page 19 of 37


Standardized coefficients Endpoint

Predictor

Estimate

SE

Z-value

P-value

Direct effects

Shredders

CAP

-0.333

0.118

-2.828

0.005

-0.662

Eucalypt

-0.333

0.118

-2.828

0.005

-0.662

Crayfish

-0.200

0.118

-1.697

0.090

-0.397

C × CAP

0.133

0.136

0.980

0.327

0.229

E × CAP

0.200

0.136

1.470

0.142

0.344

E×C

-0.067

0.136

-0.490

0.624

-0.115

CAP

-0.104

0.068

-1.530

0.126

-0.199

Eucalypt

-0.141

0.068

-2.074

0.038

-0.270

Crayfish

-0.687

0.068

-10.098

Invasive Species Mediate Insecticide Effects on Community and

Recommend Documents