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

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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

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Environmental Science & Technology 1

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

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biocide to Daphnia magna

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

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Kloasc,e

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Authors affiliations:

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a

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Germany

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

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b

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

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c

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

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d

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

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e

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

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*Corresponding author:

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Anderson Abel de Souza Machado

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Address: Müggelseedamm 310, 12587 Berlin, Germany

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Telephone: +49 (0) 30 64 181 942

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Email: [email protected]

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ACS Paragon Plus Environment

Environmental Science & Technology

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TOC Art

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Abstract

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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ecotoxicological assumption of monotonicity.

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

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Microbiological contaminant.

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1. Introduction

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

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

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

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

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

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

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

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

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

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

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agriculture and forestry.6,7,8

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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response curves using environmentally relevant concentrations of DiPel ES to aquatic

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

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

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waterborne DiPel ES over a broad range of concentrations.

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



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

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

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

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databases of the European Commission.18

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2. Experimental procedures 2.1. Physico-chemical analyses

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

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

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

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

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

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

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

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

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

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2.2. Daphnia magna exposures

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

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

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

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

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

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

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


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

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

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

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

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1

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

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

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

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

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

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

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

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

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

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neonates and 578 adults) were exposed.

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

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2.3. Biomarker measurements

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

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

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

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

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

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

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

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



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

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

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

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

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antioxidant defense and xenobiotic metabolism. Catalase was measured according to

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

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

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

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reader (Infinite M200, Männedorf, Switzerland).

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2.4. Statistical analysis

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

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

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

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

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

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

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

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

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

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

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analyses.

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

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

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


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

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

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Supporting Information.

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3. Results and Discussion

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

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

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

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

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

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

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effects of Bt pesticides on non-target organisms.7

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

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

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

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

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

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

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

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whether such currently accepted testing paradigms and government review practices are

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adequate.

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

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

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

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



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

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

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

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

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

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

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3.1. Particle size and water chemistry

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

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

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

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

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treatments.

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Light scattering analyses revealed that the various Dipel concentrations generated diverse

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

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

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

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

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

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

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


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

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

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

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

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of particles.

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

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

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

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1

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(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

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(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



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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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neonates (control = 0 ± 0 %).

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

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

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

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effective toxicity mechanism than genotoxicity might occur.

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

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

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

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

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

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

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

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

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

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

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

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

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

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monotonic effects.

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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effects.

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3.3. Multiphasic dose-responses for physiological responses

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

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

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

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

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

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

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

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

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

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

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

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determine the effects on the growth biomarker chitobiase.



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

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

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

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

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

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

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

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

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protein-1 L-1 min-1).

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

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

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

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