Low-Dose Effects: Nonmonotonic Responses for the Toxicity of a

(5, 8, 10) Thus, Bt biocides and genetically modified crops that express Bt toxins ...... A. Chitobiase of planktonic crustaceans from South Atlantic ...
0 downloads 0 Views 1MB Size
Subscriber access provided by GAZI UNIV

Article

Low-dose effects: Non-monotonic responses for the toxicity of a Bacillus thuringiensis biocide to Daphnia magna Anderson Abel Souza de Souza Machado, Christiane Zarfl, Saskia Rehse, and Werner Kloas Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b03056 • Publication Date (Web): 21 Dec 2016 Downloaded from http://pubs.acs.org on December 26, 2016

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 free 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 accessible to all readers and 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.

Environmental Science & Technology 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 27

Environmental Science & Technology 1

1

Title: Low-dose effects: Non-monotonic responses for the toxicity of a Bacillus thuringiensis

2

biocide to Daphnia magna

3 4

Authors: Anderson Abel de Souza Machadoa,b,c*, Christiane Zarfld, Saskia Rehsea,c, Werner

5

Kloasc,e

6

7

Authors affiliations:

8

a

9

Germany

Department of Biology, Chemistry and Pharmacy, Freie Universität Berlin. Berlin,

10

b

School of Geography, Queen Mary, University of London. London, UK

11

c

Leibniz-Institute of Freshwater Ecology and Inland Fisheries. Berlin, Germany

12

d

Center for Applied Geosciences, Eberhard Karls Universität Tübingen. Tübingen, Germany

13

e

Faculty of Life Sciences, Humboldt-Universität zu Berlin. Berlin, Germany

14 15

*Corresponding author:

16

Anderson Abel de Souza Machado

17

Address: Müggelseedamm 310, 12587 Berlin, Germany

18

Telephone: +49 (0) 30 64 181 942

19

Email: [email protected]

20 21 22

ACS Paragon Plus Environment

Environmental Science & Technology

Page 2 of 27 2

23

TOC Art

24 25

Abstract

26 27

Currently, there is a trend toward an increasing use of biopesticides assumed to be

28

environmentally friendly, such as Bacillus thuringiensis (Bt). Studies of the Bt toxicity to

29

non-target organisms have reported low effects at high exposure levels, which is interpreted

30

as indicating negligible risk to non-target organisms. We investigated the response of the

31

non-target organism Daphnia magna to waterborne DiPel ES, a globally used Bt formulation.

32

Neonates and adults were exposed for 48 h to a wide range of concentrations, and

33

immobilization and mortality were monitored. Whole body biomarkers (body weight, protein,

34

chitobiase, catalase, xenobiotic metabolism, and acetylcholinesterase) were measured in the

35

adults. The immobilization and mortality of the neonates were affected in a non-monotonic

36

and inverted U-shaped pattern with EC50s that were ~ 105-fold lower than those reported by

37

the manufacturer. The immobilization of adults demonstrated a similar pattern, but significant

38

mortality was not observed. The biomarker results revealed multiphasic dose-response

39

curves, which suggested toxicity mechanisms that affected various physiological pathways.

40

The main particle size in exposure media was in the size range of bacterial spores and crystal

41

toxins. However, the chemical heterogeneity was non-monotonic, with a change in the phase

42

at the maximum of toxicity (~ 5 µL L-1), which might explain the observed non-monotonic

43

effects. These results demonstrate the vulnerability of a non-target organism to a biopesticide

ACS Paragon Plus Environment

Page 3 of 27

Environmental Science & Technology 3

44

that is considered to be safe, while challenging the universal applicability of the central

45

ecotoxicological assumption of monotonicity.

46 47

Keywords: Aquatic ecotoxicology, Biomarkers, Biopesticide, Dipel, Hormesis,

48

Microbiological contaminant.

49 50

ACS Paragon Plus Environment

Environmental Science & Technology

Page 4 of 27 4

51

1. Introduction

52

53

Currently, there is a trend to replace conventional agrochemicals that have known adverse

54

side effects on environmental health with biopesticides, which are considered to be

55

environmentally friendly and safe for non-target organisms.1 In this context, products based

56

on Bacillus thuringiensis (Bt) are globally among the leading biorational insecticides2, but

57

their usage has raised some concerns regarding potentially adverse ecological effects.3,4 Bt is

58

a ubiquitous entomopathogenic Gram-positive, spore-forming bacterium that occurs naturally

59

in soils, leaves and dead insects. It synthesizes parasporal bodies with crystal endotoxins,1

60

several cytolytic proteins, exotoxins and side metabolites that act synergistically with the

61

crystal endotoxins.5 The commercial formulations of Bt are broadly used to control

62

Lepidoptera, Diptera and Coleoptera, which are vectors for human diseases as well as pests in

63

agriculture and forestry.6,7,8

64

Several tests in which non-target organisms were exposed to high levels of Bt formulations

65

did not detect deleterious effects.8, 9, 10 Exposure concentrations 2-5 orders of magnitude

66

higher than those recommended for field application often resulted in negligible effects.1

67

Additionally, the common mechanism of toxicity of Bt to target organism involves at least

68

four major steps. First, the target insect ingests Bt and/or its toxins.7 Second, enzymes

69

activate the toxins by proteolytic processing under the alkaline conditions of the midgut.11

70

Subsequently, the toxins bind to specific receptors on the gut cells.5 Finally, the toxins insert

71

through the cell membrane, which causes loss of ions and electrolytes that result in cytolysis

72

and lead to organism death.12 The requirement for this particular sequence of processes to

73

induce toxicity in target insects has been credited as the reason for the high specificity of Bt

74

insecticides.5,8,10 Thus, Bt biocides and genetically modified crops that express Bt toxins are

75

booming worldwide (Supplementary figure).13, 14, 15, 16, 17 Bt microbial pesticides represent

ACS Paragon Plus Environment

Page 5 of 27

Environmental Science & Technology 5

76

approximately 90 % of biological control agents used in the world, about 2 % of the

77

insecticides used globally, and 1 % of total pesticides.8, 9

78

Claimed to be natural and specific, Bt-based microbial pesticides have achieved notably

79

broad acceptance (Fig. 1). Dipel is a Bt formulation that is among the most-used biopesticides

80

for the control of caterpillars worldwide.10 One of its commercial forms available in Europe

81

is DiPel ES, which is presumably a mixture of Bacillus thuringiensis kurstaki (Btk), its

82

spores, crystalline endotoxins, fermentation chemicals and solids, Btk metabolites and

83

exotoxins, and formulation substances (inert and proprietary compounds). Large amounts of

84

Dipel have been sprayed over large areas of Europe, with potential exposure of aquatic

85

ecosystems. For instance, DiPel ES was applied by aerial spraying to over 185 hectares and

86

by manual processes to 5500 additional individual oaks in the forest and urban areas of

87

Frankfurt (Germany) in the year of 2015. Similar management actions are performed in many

88

other German, British and French cities (see Figure S1 in Supporting Information). However,

89

to the best of our knowledge, no detailed studies are available that have addressed dose-

90

response curves using environmentally relevant concentrations of DiPel ES to aquatic

91

organisms. Thus, we investigated the lethal and sub-lethal responses (immobilization and

92

biomarkers) in an aquatic model using the non-target organism Daphnia magna to

93

waterborne DiPel ES over a broad range of concentrations.

94 95

Figure 1: Bacillus thuringiensis (Bt) is the active compound of the most popular microbial

ACS Paragon Plus Environment

Environmental Science & Technology

Page 6 of 27 6

96

pesticides. The U.S. data refer to all monitored Bt subspecies17. For the Brazilian data, the

97

numbers from Brazilian authorities were extrapolated from the scale of states to biomes by

98

the authors. The data for the European countries were compiled in 2015 from public

99

databases of the European Commission.18

100

101

102

2. Experimental procedures 2.1. Physico-chemical analyses

103

Physico-chemical measurements were performed in duplicate. The chemical behavior of

104

DiPel ES in the medium that was used to expose the daphnia was analyzed in terms of

105

chemical heterogeneity (polydispersity index) and the modes and importance of particle size

106

distribution using light-scattering measurements (22 ˚C, scattering angle 173˚) with the

107

Zetasizer nano ZSP (Malvern, Worcestershire, UK). These measurements were not stable

108

below 0.1 µL DiPel ES L-1, therefore only higher concentrations were considered for the light

109

scattering analysis. The carbon concentration was additionally measured with a C/N-

110

Analyzer (TOC 5000, Shimadzu, Kyoto, Japan), which had a detection limit of 1 mg C/N L-1.

111

Basic water chemistry parameters (pH, dissolved oxygen) were also determined.

112

2.2. Daphnia magna exposures

113

A D. magna culture originating from a female from Lake Großer Müggelsee (Berlin,

114

Germany) has been maintained in the Ecophysiology Laboratory of the Leibniz-Institute of

115

Freshwater Ecology and Inland Fisheries for ~ 7 years.19 DiPel ES (Cheminova Deutschland

116

GmbH & Co. KG; Valent BioSciences, Libertyville-U.S.), hereafter referred to as Dipel,

117

contains the Btk ABTS 351 HD-1 and was obtained as a sample of the product that was

118

recently sprayed over the state of Brandenburg (Germany). Neonates (< 24 h old) of D.

119

magna were exposed for 48 h to waterborne Dipel at 24 different Dipel concentrations

ACS Paragon Plus Environment

Page 7 of 27

Environmental Science & Technology 7

120

(0.0025 to 320 µL L-1) using 3-6 replicates, each of which consisted of 10 mL of ISO test

121

water with ~ 5 neonates (each in 20 mL glass flasks), as well as the controls according to

122

OECD guidelines.20 Adult females of D. magna (17-21 days-old) born in the same period as

123

the neonates were exposed in the same test water to 9 Dipel concentrations (0.01 to 500 µL L-

124

1

125

was repeated 5 months later to confirm the reproducibility of the dose-response pattern and to

126

provide enough material for the biochemical analyses. The daphnids were evaluated after 24

127

h and 48 h of exposure for immobilization and mortality. Animals unable to swim within 15

128

seconds after gentle agitation of the test vessel were considered to be immobilized.20

129

Mortality was assumed if a complete absence of macroscopic movement was observed during

130

the same 15 s period. Dead daphnia were an opaque white color that confirmed absence of

131

life-sustaining functions. After exposure, the neonates were discarded, whereas each adult

132

was quickly and gently dried in paper napkins, weighed, and preserved individually in bullet

133

tubes at -70 ˚C for the subsequent biochemical analyses. Totals of 1,145 daphnia (567

134

neonates and 578 adults) were exposed.

, 4-6 replicates, each consisting of 50 mL and ~ 4 adults) plus controls. The adult exposure

135 136

2.3. Biomarker measurements

137

The adults from both exposures were used for biomarker analyses. The whole body of each

138

animal was homogenized in 200 µL of cold phosphate buffer (0.1 M, pH 7.5) for 1.5 min. at

139

18 cycles s-1 using TissueLyser (Qiagen-Retsch Stokcach, Germany). The biomarkers were

140

measured in these homogenates. The number of animals used for each biomarker per

141

treatment (N) varied according to our experience on the biomarker variance as well as to the

142

amount of tissue required and available. The total protein in these homogenates was

143

measured using a Bradford assay kit (N = 18-23, Sigma Aldrich, Germany). Chitobiase

144

activity, a biomarker for crustacean growth, was measured according to Avila et al.21 with the

ACS Paragon Plus Environment

Environmental Science & Technology

Page 8 of 27 8

145

modification that phosphate buffer (0.1 M, pH 7.0) was used as the reaction media (N = 10-

146

12). Acetylcholinesterase activity was measured according to Ellman et al.22 as a biomarker

147

for neurotransmission (N = 4-9). Finally, catalase (N = 10-12), glutathione S-transferase (N =

148

4-6), and glutathione reductase (N = 5-6) activities were analyzed as biomarkers for

149

antioxidant defense and xenobiotic metabolism. Catalase was measured according to

150

Beutler,23 while glutathione S-transferase and glutathione reductase were estimated according

151

to Keen et al.24, and Carlberg and Mannervik,25 respectively. All assays were adapted to use

152

96 well-microplates in which the absorbance or fluorescence was read using a Tecan plate

153

reader (Infinite M200, Männedorf, Switzerland).

154

155

2.4. Statistical analysis

156

The Trimmed Spearman-Karber method was used to estimate LC50 (mortality) and EC50

157

(immobilization)26 using TSK software. This method is recommended by the Environmental

158

Protection Agency (U.S. EPA)26 and is among the most common methods used to estimate

159

LC50 and EC50. For the determination of the LC50 and EC50 values, a subset of the test

160

concentration had to be used because the TSK method requires monotonicity and limits the

161

number of exposure concentrations to a maximum of 10. Therefore, estimation of the LC50

162

and EC50 values for the neonates was based on concentrations up to 5 µL Dipel L-1, whereas

163

for adults concentrations up to 10 µL Dipel L-1 were chosen (see details in the Supporting

164

Information). Selection of data was a requirement for the method and did not affect p values

165

presented here. Additionally, no selection of data was performed for any other statistical

166

analyses.

167

Significant differences in the mortality and immobilization were detected using the Fisher

168

test,27 and differences in the biomarkers were detected using the Kruskal-Nemenyi test with

169

Tukey post hoc test for the complete data set.28 Linear correlations between the Dipel

ACS Paragon Plus Environment

Page 9 of 27

Environmental Science & Technology 9

170

concentrations and biomarkers were also tested in the complete data set.27 For all analyses,

171

the significance level was 5 % (α = 0.05), and all data discussed here are available in the

172

Supporting Information.

173

174

3. Results and Discussion

175

Bt sprays such as Dipel are applied several times in a growing season to reach the entire

176

larval pest population, which results in considerable amounts of total deposition.10 Other Bt

177

products, i.e., those based on Bti, are applied directly into water environments, which

178

increases the risk of exposure to non-target aquatic biota. Both Bt toxins and spores have the

179

potential for indirect ecological side-effects2,3 because they persist for weeks to years in lentic

180

and lotic environments.5,11 Nonetheless, little scientific attention has been given to the direct

181

effects of Bt pesticides on non-target organisms.7

182

Concomitantly, there is growing discussion regarding the relevance of non-monotonic

183

ecotoxicological responses.29 Some studies have suggested that a central ecotoxicological

184

principle, i.e., that toxicity increases monotonically with the exposure levels, might not be

185

universally correct.30 These authors have argued that non-monotonicity has been generally

186

neglected by ecotoxicology due to constraints of experimental design and lack of proper

187

dose-response curves. Likewise, environmental agencies,31 the European Commission and

188

agencies,32 and several American scientific societies33 have expressed concern with respect to

189

whether such currently accepted testing paradigms and government review practices are

190

adequate.

191

Therefore, the present results are scientifically and socially relevant for two main reasons.

192

First, they show the potentially high toxicity of a biopesticide that has been assumed to be

193

safe to a relevant non-target ecotoxicological model organism. Second, the present results

194

report unprecedentedly unusual non-monotonic dose-responses. In the next paragraphs, we

ACS Paragon Plus Environment

Environmental Science & Technology

Page 10 of 27 10

195

present the results from Dipel chemical behavior in the exposure media. Next, we explore the

196

inverse U-shaped dose-response for the organism toxicity and how it relates to Dipel

197

behavior. Then, we address the multiphasic responses of the physiological biomarkers.

198

Finally, we discuss the implications of these observations for environmental health

199

regulation. Investigations of the direct or indirect effects of single components of Dipel

200

mixture (e.g., Bt cells, Bt spores, Bt toxins) were beyond the scope of the current study.

201

202

3.1. Particle size and water chemistry

203

Dissolved oxygen and pH were relatively constant over the range of concentrations tested

204

(8.71 ± 0.01 mg O2 L-1 and 7.74 ± 0.01, respectively). Organic carbon could be detected but

205

not quantified at 500 µL Dipel L-1; therefore, it complied with the OECD criteria (total

206

organic carbon < 2 mg L-1, total particulate solids < 20 mg L-1) 20 in all experimental

207

treatments.

208

Light scattering analyses revealed that the various Dipel concentrations generated diverse

209

particle size distributions in the exposure media (Fig. 2). The heterogeneity of the particle

210

sizes in the exposure media (as indicated by the polydispersity index) decreased with

211

increasing Dipel concentrations up to ~ 5 µL L-1 (Fig. 2A) and increased at concentrations

212

above that level. Particles in the size range of bacteria spores and crystal endotoxins (~ 100-

213

300 nm) predominated, whereas smaller and larger particles were observed at the lowest and

214

highest concentrations (Fig. 2B). This generated a bimodal distribution with two particle size

215

modes in the exposure media (Fig. 2C).

ACS Paragon Plus Environment

Page 11 of 27

Environmental Science & Technology 11

216 217

Figure 2: Dipel behavior at various concentrations (each point represents an individual

218

measurement). A: The triangles represent the polydispersity index. B: Main mode (black-

219

filled circles) and secondary mode (white-filled circles) of the particle sizes in the exposure

220

media. C: Importance of main (black-filled circles) and secondary mode (white-filled circles)

221

of particles.

222

3.2. Inverted U-shaped dose-response for organism level responses

223

224

Dipel affected the immobilization and mortality of the neonates in an inverted U-shaped

225

dose-response curve (p < 0.001, Fig. 3). For the concentrations less than and equal to 5 µL L-

226

1

227

(0.130- 0.168) µL Dipel L-1, LC50,24h= 0.880 (0.595- 1.302) µL Dipel L-1, and LC50,48h= 0.286

228

(0.238- 0.342) µL Dipel L-1.

, the toxicity levels were EC50,24h= 0.148 (0.129- 0.171) µL Dipel L-1, EC50,48h= 0.148

ACS Paragon Plus Environment

Environmental Science & Technology

Page 12 of 27 12

229 230

Figure 3: Organism-level toxicity of Dipel to Daphnia magna neonates (average ± SEM, 4

231

replicates per treatment, 14 replicates of the controls, N = 5). The circles indicate values that

232

were not significantly different from the control. The triangles indicate values that were

233

significantly different from the control. The red-filled symbols indicate treatments with

234

averages higher than the control average ± SEM, and the green-filled symbols indicate

235

treatments within the control ± SEM. A: Immobilization at 24 h of exposure (control = 3 ± 2

236

%); B: Immobilization at 48 h of exposure (control = 6 ± 4 %); C: Mortality at 24 h of

237

exposure (control= 0 ± 1 %); D: Mortality at 48 h of exposure (control= 0 ± 1 %).

238

239

Immobilization of the adults was also affected by Dipel (p < 0.001). The EC50 values for the

240

adults exposed simultaneously with the neonates were EC50,24h= 0.949 (0.735 - 1.225) µL

241

Dipel L-1, EC50,48h= 0.292 (0.194 - 0.441) µL Dipel L-1. The adults exposed 5 months later

242

demonstrated a similar response pattern (EC50,24h= 0.175 (0.081 - 0.378) µL Dipel L-1,

243

EC50,48h= 0.143 (0.076 - 0.271) µL Dipel L-1) (Fig. 4): the effects on mortality were non-

244

significant in adults. The immobilization and mortality decreased at concentrations greater

245

than 10 µL Dipel L-1 and generally disappeared at concentrations higher than 90 µL Dipel L-1

ACS Paragon Plus Environment

Page 13 of 27

Environmental Science & Technology 13

246

for adults and neonates. The acute EC50 values presented here are ~ 105-fold lower than the

247

chronic concentrations indicated by the Dipel manufacturer (EC50,32days: 14 mg L-1). Indeed,

248

given these results, Btk in Dipel formulation seems to be more toxic to D. magna than

249

Bacillus thuringiensis israelensis (Bti) to the target organism Aedes vexans.6

250 251

Figure 4: Organism-level toxicity of DiPel ES to Daphnia magna adults (average ± SEM, 7-

252

12 replicates per treatment, 20 replicates of controls, N = 3). The circles indicate values that

253

were not significantly different from the control. The triangles indicate values that were

254

significantly different from the control. The red-filled symbols indicate treatments with

255

averages higher than the control average ± SEM, and the green-filled symbols signify

256

treatments within the control ± SEM. A: Immobilization at 24 h of adults exposed at the same

257

time as neonates (control = 0 ± 0 %); B: Immobilization at 48 h of adults exposed at the same

258

time as neonates (control = 0 ± 0 %); C: Mortality at 48 h of adults exposed at the same time

259

as neonates (control = 0 ± 0 %); D: Immobilization at 24 h of adults exposed 5 months later

260

than neonates (control = 0 ± 0 %); E: Immobilization at 48 h of adults exposed 5 months later

261

than neonates (control = 0 ± 0 %);F: Mortality at 48 h of adults exposed 5 months later than

262

neonates (control = 0 ± 0 %).

263

ACS Paragon Plus Environment

Environmental Science & Technology

Page 14 of 27 14

264

Such inverse U-shaped toxicity at organismal level is rather unusual. It has been observed

265

mostly for the chronic toxicity caused by carcinogenics and endocrine disruptors.29,34 On the

266

basis that the neonates were immobilized within a few minutes after exposure, a more acutely

267

effective toxicity mechanism than genotoxicity might occur.

268

Bt toxins cause cytotoxicity, ionic disruption, and osmolyte loss in vertebrate and invertebrate

269

cell cultures.5,11,35 However, such effects remain to be demonstrated in vivo in non-target

270

organisms. Additionally, the pH in the digestive tract of D. magna ranges from 6 to 7.2, at

271

which activation of the endotoxin crystals is unlikely.5,11 Thus, it is possible that other Bt or

272

Dipel-related stressors are responsible for the toxicity to D. magna.

273

In this context, the chemical heterogeneity varied as a function of the Dipel concentration in

274

an U-shaped fashion. The particle size present throughout the exposure concentrations was

275

100-300 nm in diameter, in the range of the sizes of both the Bt parasporal inclusions (crystal

276

endotoxins) and Bt spores. At the highest concentrations, additional larger particle sizes were

277

observed. This is attributable to the higher instability in the solubility of Dipel colloids, where

278

large aggregates could potentially encapsulate toxic compounds, which would reduce the

279

bioavailability. Therefore, interactions between the chemical behavior at the various

280

concentrations of Dipel and the physiology of daphnids might explain the observed non-

281

monotonic effects.

282

These results challenge the idea that low toxicity at high exposure implies lower or no

283

toxicity at lower concentrations. The current standard ecotoxicological techniques could not

284

determine LC50 and EC50 values based on the full data set due to the clearly biphasic and

285

inverted U-shaped response. Hence, only the low concentrations were used to determine these

286

parameters because otherwise two EC50s could be derived, i.e., when toxicity is increasing or

287

decreasing. Similarly, multiple no-observed effect concentrations (NOEC) exist. Finally, in

288

addition to the lowest observed effect concentration (LOEC), it is necessary to conceptualize

ACS Paragon Plus Environment

Page 15 of 27

Environmental Science & Technology 15

289

a maximum observed effect concentration (MOEC), which in the present study was ~ 80 µL

290

L-1. Concentrations above MOEC yielded no detectable effects.

291

It is worth mentioning that without the monotonicity assumption, NOECs, LOECs and

292

MOECs are properties of the experimental design and not of the toxicant. In our experiments,

293

the exposure limit was 320 µL L-1 for neonates and 500 µL L-1 for adults. Above this range,

294

turbidity prevented observation of the organisms and classification of swimming ability.

295

Presumably, concentrations much higher than the observed MOEC would cause further

296

effects.

297

3.3. Multiphasic dose-responses for physiological responses

298

Effects of Dipel were observed for most biomarkers, and the differences are stronger when

299

compared among treatments than with the controls. Dipel exposure affected the body weight

300

and chitobiase activity of D. magna (p < 0.01, Fig. 5). There was a trend for an increase in

301

the body weight with exposures higher than 1 µL Dipel L-1 (r2 = 0.17, p < 0.001). There were

302

no significant changes in the total protein. Despite the significant effects observed for the

303

chitobiase activity, none of the tested concentrations was different from the control, i.e., the

304

differences were only significant among the treated groups. Feeding of the exposed

305

organisms on Bt might explain the effects on body weight, i.e., the digestive tracts of

306

daphnids exposed to high concentrations were filled. Indeed, D. magna feeds on particles

307

from 1-50 µm, which include the sizes of bacteria and Bt spores. In turn, the balance between

308

the energy obtained from food and the metabolic costs of Dipel detoxification could

309

determine the effects on the growth biomarker chitobiase.

ACS Paragon Plus Environment

Environmental Science & Technology

Page 16 of 27 16

310 311

Figure 5: General health biomarkers on Daphnia magna adults after a 48 h Dipel exposure

312

(average ± SEM). The circles indicate values that were not significantly different from

313

control. The triangles indicate values that were significantly different from the control. The

314

red-filled symbols indicate treatments with averages higher than the control average ± SEM,

315

the blue-filled symbols indicate treatments with averages lower than the control ±SEM, and

316

the green-filled symbols indicate treatments within the control ± SEM. A: Body weight (N =

317

21 ± 1, control = 1.45 ± 0.08 mg); B: Body composition (N = 21 ± 1, control = 6.6 ± 0.5 mg

318

protein g wt-1); C: Chitobiase activity (N = 12 ± 0, control = 41.82 ± 1.78 µmol MUF mg

319

protein-1 L-1 min-1).

320

321

Catalase, glutathione S-transferase, and glutathione reductase also demonstrated non-

322

monotonic and multiphasic responses. Catalase (p < 0.01), glutathione S-transferase (p