Daphnid Life Cycle Responses to the Insecticide Chlorantraniliprole

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Daphnid life cycle responses to the insecticide chlorantraniliprole and its transformation products Vesna Lavtizar, Rick Helmus, Stefan Kools, Darko Dolenc, C.A.M. van Gestel, Polonca Trebše, Susanne Waaijers, and Michiel Kraak Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es506007q • Publication Date (Web): 17 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Environmental Science & Technology

Daphnid life cycle responses to the insecticide chlorantraniliprole and its transformation products

3 4

VesnaLavtižara*, Rick Helmusb, Stefan A.E. Koolsc, DarkoDolencd, Cornelis

5

A.M. van Gestele, PoloncaTrebšea, f, Susanne L. Waaijersg,Michiel H.S. Kraakb

6

a

7

301, 5000 Nova Gorica, Slovenia

Laboratory for Environmental Research, University of Nova Gorica, Vipavska 13, P.O. Box

8 9 10

b

Institute for Biodiversity and Ecosystem Dynamics (IBED), University of Amsterdam, P.O. Box

94248, 1090 GE Amsterdam, The Netherlands

11 12

c

KWR Research Institute, P.O. Box 1072, 3430 BB Nieuwegein, The Netherlands

13 14 15

d

Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, 1000

Ljubljana, Slovenia

16 17 18

e

Department of Ecological Science, Faculty of Earth and Life Sciences, VU University

Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands

19 20

f

Faculty of Health Sciences, University of Ljubljana, Zdravstvena pot 5, 1000 Ljubljana, Slovenia

21 22

g

RIVM, Postbus 1, 3720 BA Bilthoven, The Netherlands

23 24

*Corresponding author: VesnaLavtižar ([email protected]),+386 41 278 567

25

Abstract 1 ACS Paragon Plus Environment


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Chlorantraniliprole (CAP) is a newly developed, widely applied insecticide. In the

27

aquatic environment, several transformation products are formed under natural

28

conditions, one by dehydration and others by photo-induced degradation. Data on

29

aquatic ecotoxicity of CAP can mainly be found in registration and regulatory

30

evaluation reports. Moreover, the toxicity of its transformation products and

31

especially effects upon chronic exposure remain completely unknown. Hence, our

32

aim was to investigate the acute and chronic toxicity of CAP and its transformation

33

products to the daphnid Daphnia magna. The results showed that CAP is extremely

34

toxic to D. magna, with an acute and chronic LC50 of 9.4 and 3.7 µg/L,

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respectively. No effects on daphnid reproduction were observed, but the impact on

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daphnid survival also affected population growth rate, with an EC50 of 3.5 µg/L. In

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contrast, no negative effects of the two main degradation products were observed.

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The present study demonstrated a high sensitivity of non-target microcrustaceans to

39

CAP. However, the actual risk of CAP in water diminishes with its spontaneous or

40

light-induced degradation into two transformation products, showing no toxicity to

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the daphnids in the present study.

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43

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Introduction

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Large-scale application of pesticides still is the main method for crop protection in

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modern agriculture. Because of the environmental hazard of previously used

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pesticides, there is an ongoing need to develop new, less hazardous insecticides.

48

One of these new insecticides is chlorantraniliprole (CAP) (Figure 1, left),

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belonging to the antranilicdiamides. Positive experiences with CAP for pest

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control, especially due to its high insecticidal activity, made CAP widely used. Its

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formulated products became registered in many countries1 and are allowed for

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treating an increasing number of crop species.2 CAP is exceptionally active against

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a large range of insect pests and is highly selective.3, 4, 5 It binds to the ryanodine

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receptors in insects, causing an unregulated release of Ca2+ from the intracellular

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calcium deposit stores, resulting in permanent muscle contraction and death of the

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insect.4,

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expressing ryanodine receptors revealed that CAP features a high toxicity towards

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insects (EC50 of 0.04 µM for the ryanodine receptors in the fruit fly Drosophila

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melanogaster), but a very low toxicity to mammalian ryanodine receptors (EC50 of

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14 µM for the mouse myoblastcell line C2C12).3

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The high selectivity and toxicity of CAP towards insects raised concerns about the

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effect on non-target insects and other arthropods, but studies on the toxicity of CAP

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to non-target organisms are scarce. Bruggeret al.6 summarized the research on the

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effects of CAP, the technical product as well as its formulated products, on seven

5

Comparing the toxicity of CAP to insects and mammalian cell lines



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species of parasitic wasps. In the 24-h acute tests, no effect was observed applying

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the worst case scenarios, testing concentrations above crop-relevant exposure

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levels. Little or no effect was also observed on bumblebees,

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several soil invertebrates in a field experiment.9

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According to the US EPA10 the lethal concentrations for selected freshwater fishes

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are in all cases above the CAP solubility in water, however this data concerns

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mainly standard acute, 96-h short-term exposures (OECD 203)11. An LC50of 14,400

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µg/L was reported for the fresh water fish Channa punctatus12 in a 96-h exposure

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test and a 96-h LC50 of 11,000 µg/L was reported for the grass carp

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Ctenopharingodon idella13. However, the values of the latter two studies most

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likely correspond to the concentrations tested with the CAP formulated product

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(18.5% SC), and not to CAP as an active ingredient. In contrast, EPA10 and

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EFSA14,

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mayfly

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caddisflyChimarraatterima(LC50=

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Chironomusriparius(LC50 = 85.9 µg CAP/L), the water flea Daphnia magna (LC50

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= 11.6 µg CAP/L) and the amphipod Gammaruspseudolimnaeus(LC50 = 35.1 µg

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CAP/L). Among the estuarine and marine invertebrates, EPA10 and EFSA14,

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report high toxicity of CAP to the eastern oyster Crassostreavirginicawithan acute

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EC50 of 39.9 µg CAP/L. CAP is moderately toxic to the crayfish Oronectesvirilis

15

7

honeybees8 and

reported very high acute toxicity to aquatic invertebratessuch as the Centroptilumtriangulifer(LC50 11.7

=11.6 µg


µg

CAP/L),

CAP/L),the

the midge

15

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(48-h LC50> 1420 µg/L, NOEC = 759 µg/L)10, 14, 15 and highly toxic (LC50 = 951

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µg/L) to the crayfish Procambarusclarkii in an acute (96 h) toxicity test.16

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Although it is beneficial that modern insecticides are less persistent than their

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precursors, this also raised concerns about the possible toxicity of unknown

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transformation products. Sometimes these transformation products appeared to be

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more toxic than their parent compounds or exerted a different mode of action (see

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for example17,

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showed that, in the absence of light, it transforms spontaneously by dehydration

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into the stable product 2-(3-bromo-1-(3-chloropyridin-2-yl)-1H-pyrazol-5-yl)-6-

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chloro-3,8-dimethylquinazolin-4(3H)-one (see Figure 1, transformation product 1

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(TP1), middle).20 Degradation of CAP is highly dependent on the pH of the

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medium. Stability experiments showed that CAP is stable at acidic pH, but tends to

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degrade when pH is above neutral.20 The half-life of CAP (initial concentration =

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0.6 µg/L) in buffered water of pH 9 was 10 days.21 Moreover, CAP is also

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susceptible to photodegradation: under UVA it degrades into the compound 2-(3-

18, 19

). In natural water, CAP is indeed not very stable, since we

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bromo-1-(3-hydroxypyridin-2-yl)-1H-pyrazol-5-yl)-6-chloro-3,8-

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dimethylquinazolin-4(3H)-one (Figure 1, transformation product 2 (TP2), right).20

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Both degradation products are assumed to be the main transformation products of

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CAP in natural waters. It is thus expected that aquatic organisms may be exposed

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both to CAP and to its transformation products. While high toxicity of CAP to non5 ACS Paragon Plus Environment


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target aquatic invertebrates has been reported10, 14, 15, the effects of the degradation

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products as well as their chronic toxicity remain completely unknown. Hence, our

107

aim was to investigate the acute and chronic toxicity of CAP and its transformation

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products to the daphnid Daphnia magna. This allowed a more reliable

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environmental risk assessment of the compounds,22 as the concentrations of the

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compounds in natural waters are assumed to be low and effects might be expressed

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over a longer period of time.

112 113

Materials and methods

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Test organism

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We selected the fresh water crustacean Daphnia magna Straus to test the acute and

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chronic toxicity of CAP and its transformation products TP1 and TP2. Daphnids

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play a significant ecological role in freshwater food webs23 and D. magna is

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frequently used as a test organism in aquatic ecotoxicity studies because of its

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parthenogenetic reproduction, short life cycle, high fecundity and ease of

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culturing,24 with several standardized test guidelines currently available.25, 26, 27

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D. magna neonates (younger than 24 h, clone 4) were obtained from

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GrontmijAquasense (Amsterdam, the Netherlands), where they were cultured as

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described by Waaijers et al.28 Acute toxicity tests with the reference compound 6 ACS Paragon Plus Environment

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K2Cr2O7 were performed on a regular basis, in order to check whether the

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sensitivity of D. magna culture was within the limits (EC50 24-h = 0.6-2.1 mg/L), as

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set by the guideline.25

128 129

Test compounds

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An analytical standard of chlorantraniliprole of 99.5% purity, used for the toxicity

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tests and for the chemical analysis, was purchased from Dr. Ehrenstorfer GmbH,

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Augsburg, Germany. Since the transformation products are not commercially

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available, we synthesized TP1 and TP2 ourselves (University of Nova Gorica,

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University of Ljubljana, Slovenia), as described by Lavtižaret al.20 The purity of the

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transformation products was >99% for TP1 and >97% for TP2 (for chemical

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structures, see Figure 1).

137 138

Toxicity tests

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Acute toxicity tests

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To determine the acute toxicity of CAP and its transformation products, daphnids

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were exposed in 48-h immobility tests, according to OECD guideline 202,26 except

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where noted. The previously reported acute EC50 value10 served to select the

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concentration range for CAP. Only one concentration was tested for TP1 (0.14

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mg/L), which represents the limit of its water solubility.15 The maximal tested 7 ACS Paragon Plus Environment


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concentration for TP2 was chosen according to the maximal dissolved

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concentration of CAP (0.88 mg/L) in water.10, 21 In a natural aquatic system, it is

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not expected to measure concentrations of TP2 higher than those of CAP. The

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following nominal concentrations were tested: 0, 2, 5, 10, 20, 50 µg/L CAP; 0,

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0.05, 0.1, 0.2, 0.5 and 1 mg/L TP2 and 0.14 mg/L for TP1. All solutions were

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prepared in ISO medium.26 Because of the low solubility of the compounds in

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water (CAP: 0.88 mg/L10,

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solvent and therefore a solvent control was also included. DMSO was chosen due

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to its low toxicity to D. magna compared to other organic solvents often employed

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in toxicity tests.29Firstly, the stock solution of each compound was prepared in

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DMSO. From this stock solution, the final concentrations in ISO medium were

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prepared. Since different concentrations were tested for each compound, different

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volumes of DMSO from the compound stock solution were added to the medium.

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A concentration of 0.0006 v/v % of DMSO was used in the tests with CAP and TP1

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and 0.0024 v/v % for TP2, with all treatments per toxicity test containing the same

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solvent concentration. The higher volume of DMSO contained in the test solutions

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with TP1 is due to different concentration of the stock solutions, from which the

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final concentrations of the test solutions were made.

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Per test concentration, four replicates were prepared. Each replicate consisted of a

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polypropylene tube containing 40 mL of test solution into which five daphnid

21

) dimethysulfoxide (DMSO) was used as a carrier


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neonates, younger than 24 h were placed, using a disposable transfer pipette. The

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test tubes were randomly distributed in a climate controlled fume hood (20 ± 1ºC),

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with a 16:8 h light-dark regime.

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After 24 and 48 h the daphnids were checked for immobility. The daphnids that

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were not able to swim after a gentle stimulation by tapping the tubes, were

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considered immobilized. Physical-chemical parameters (temperature, oxygen level,

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pH, hardness and conductivity) were determined at the beginning and at the end of

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test for all test concentration, as recommended by the guideline.26For chemical

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analysis of the actual test concentrations, see SI4.

174

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Chronic toxicity tests

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To determine the chronic toxicity of CAP and its transformation products, 21-day

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daphnid reproduction tests were performed following OECD guideline 211,25

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except where noted. Nominal test concentrations were chosen according to the

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results obtained from the acute tests, and were: 0, 1, 3, 6, 9, 12 µg/L CAP; 0, 0.05,

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0.1, 0.2, 0.5 and 1 mg/L TP2 and 0.14 mg/L TP1. Test solutions were prepared in

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Elendt M4 medium25 and DMSO was used as a carrier solvent (0.00012 % (v/v) in

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the test with CAP and TP1 and 0.0025 % (v/v) in the test with TP2. Per test

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concentration, fifteen replicates were prepared. Each replicate consisted of a 50 mL

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polypropylene tube containing 40 mL of test solution. The tubes were randomly 9 ACS Paragon Plus Environment


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distributed in a controlled fume hood (20 ±1ºC) with a light-dark regime of 16:8 h.

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The experiment was started by introducing one daphnid neonate younger than 24 h

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into each test vessel. The test solutions were renewed three times a week (Monday,

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Wednesday, Friday) using freshly prepared stock solutions. Before and directly

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after renewal, oxygen concentration, temperature and pH were measured, as

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recommended by the guideline.25For chemical analysis of the actual test

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concentrations, see SI4. Daphnids were fed daily with a concentrated algae

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suspension

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Amsterdam. The density of algal suspension was 2850 cells/µL28 and the daily

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aliquots per daphnid were: day 0-2: 450 µL, day 3-5: 700 µL, and days 6-21: 900

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µL. Daily, the daphnids were checked for immobility and mortality was recorded if

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no movement was noticed after a gentle stimulus. When reproduction started, the

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offspring was counted and removed from the vessels on a daily basis.

(Scenedesmussubspicatus)

obtained

from

GrontmijAquasense,

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Data analysis

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Data for survival were first tested for normality applying the D'Agostino-

201

Pearsonomnibus K2 normality test. Controls and solvent controls were compared

202

using an unpaired Student’s t-test. When they did not differ significantly, the

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controls were pooled. The different treatments within the test were tested for


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significance using one-way analysis of variance (ANOVA), followed by Tukey's

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multiple comparison post hoc test. To compare treatments with the controls,

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Dunnett's multiple comparison test was used.

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Grubbs’ test was used to detect possible outliers among the replicates. Significant

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outliers were removed when P < 0.05.

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Concentration-response relationships were calculated according to Haanstra et al.,30

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in which the data of the toxicity end point were plotted against the actual

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concentration of CAP in the test solution by fitting a logistic curve (Equation 1).

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() =

213

( )

(Eq. 1)

214 215

Here, the y(x) is the response of the end point (immobility) at concentration x, a is

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the EC50 (µg/L), b stands for the slope of the curve, c is the average mobility of the

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control (y(0)), and x is the concentration of CAP in the water (µg/L).

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In the chronic toxicity tests, fecundity was examined as an additional toxicity end

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point, expressed as cumulative reproductive output (CRO). CRO was calculated as

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follows (Equation 2):

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= ∑

222

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(Eq.2 )

223 224

where is the cumulative reproductive output per surviving parent animal for a

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specific treatment (control or exposure concentration), is the time of experiment

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in days with Ω as the last day of the experiment (21 days) and is the number of

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living offspring per adult at time .

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data set was first tested for normality using the D'Agostino-Pearsonomnibus

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K2 normality test. ANOVA was applied to compare the data between different

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treatments for each compound and their corresponding controls followed by

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Dunnett's Multiple Comparison post hoc test.

232 233

The population growth rate (r) was calculated from the integration of the life

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history data on survival probability and fecundity, using the Lotka-Euler equation

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(Equation 3).31, 32

236 237

"#( ) 1 = ∑ !

(Eq. 3),

238 239

where t is the time of the experiment (days), lasting for Ω (21) days, lt is survival

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probability at time t, mt is the number of living neonates produced per adult at time

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t and $ is the population growth rate (day-1). Population growth rate ($) and its error 12 ACS Paragon Plus Environment

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were calculated with the open source program R (version 3.1.1. for Windows),

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where Kaplan-Meier survivorship analysis was used.33

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All statistical analyses were performed using GraphPad Prism 5.03, while the

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population growth rate and its standard error were calculated using the software

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program R, following the script developed by Arne Jansen, University of

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Amsterdam. For this, a Jackknife method was used.34

248

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Results

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Acute toxicity tests

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The sensitivity of the daphnids to the reference toxicant K2Cr2O7 (24-h EC50 = 1.1

252

mg/L, 95% CI: 0.8-1.3 mg/L) was within the prescribed range (24-h EC50 = 0.6-2.1

253

mg/L) as set by the guideline.25 Also the physical-chemical parameters (hardness,

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oxygen level, temperature and pH) of the test solutions were within the

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recommended ranges.25The control and solvent control did not significantly (P >

256

0.05) differ from each other and were therefore pooled. The mean control survival

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was 93 % or higher, which meets the validity criteria (survival of the controls over

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90 %), set by OECD guideline 202.25The transformation product TP1 showed no

259

effect on the daphnids, while TP2 caused some immobility (35 %) at the two

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highest test concentrations (1.2 and 0.6 mg/L). In contrast, for the parent compound 13 ACS Paragon Plus Environment


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CAP, a clear concentration-response relationship was observed (Figure 2), from

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which an EC50 value of 9.4 µg/L (95% CI: 9.1-9.6) was derived.

263

264

Chronic toxicity tests

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The physical-chemical parameters of the test solutions (temperature, pH, hardness

266

and oxygen concentration) were within the recommended ranges.25In all tests, the

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control survival was 93-100 %. In the chronic toxicity test, TP1 had no effect on

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survival nor on reproduction of the daphnids, confirming the lack of effect

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observed in the acute test. In contrast to the slight mortality observed in the acute

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test at the highest test concentration, during chronic exposure no significant (P >

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0.05) effect on survival was observed for TP2 at the same test concentrations. The

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cumulative reproductive output after 21 days exposed to TP2 was comparable to

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that of the controls, except for the highest test concentration (0.9 mg/L), where

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reproduction was even slightly stimulated, although the difference compared to the

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corresponding control was not significant (P > 0.05).

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There was also no significant difference (P > 0.05) in the age at first reproduction

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between controls, TP1 (0.14 mg/L, nominal) and all tested concentrations of TP2.

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In contrast to the two transformation products, the parent compound CAP clearly

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affected daphnid survival at concentrations of 8.0 µg/L and higher, all causing


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complete mortality within 4 days (Figure 3, left panel). From the survival data at

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the end of the experiment (21 d) a clear concentration–response relationship was

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obtained (Figure 3, right panel), from which an LC50 value of 3.7 µg/L (95% CI:

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3.2-4.2) was derived. The comparison between the acute and chronic concentration

284

response curves demonstrates that the toxicity of CAP increases with increasing

285

exposure time with an acute to chronic ratio (ACR) of 2.5.

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While CAP showed a clear effect on the survival of the exposed daphnids, no effect

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on reproduction of the surviving adults was observed, as the cumulative

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reproductive output as well as the age at first reproduction between CAP

289

concentrations and corresponding controls did not significantly differ (P > 0.05).

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As CAP severely affected the survival of the daphnids, also the population growth

291

rate was affected with increasing CAP concentrations, resulting in the

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concentration–response relationship plotted in Figure 4. From this relationship, an

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EC50 for the effect on population growth rate was derived, being 3.5 µg/L (95% CI:

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3.4-3.6).

295

296

DISCUSSION

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The present study showed that the two transformation products elicited hardly any

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effect on the daphnids, whereas CAP appeared to be highly toxic to D. magna. The

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acute EC50 for CAP (9.4 µg/L) obtained in our study is similar to the value 15 ACS Paragon Plus Environment


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previously reported by the EPA10 and EFSA14,

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measured concentrations and immobility.

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The immobilization EC50 for CAP in the chronic test was 2.5 times lower than in

303

the acute test. When daphnids were exposed to the highest CAP concentrations

304

(10.8, 9.5 and 8.0 µg/L) a significant effect on mobility was observed already after

305

2 days of exposure. Although sublethal effects (such as reproduction impairments,

306

reduced body size, production of winter eggs) are often expressed in animals

307

chronically exposed to toxicants, here immobilization was the only effect observed

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with CAP. One reason for this could be a fast degradation of CAP in water into less

309

or non-toxic degradation products. In this case, the compound would acutely affect

310

survival, but because of its fast degradation, long-term effects on reproduction

311

would not be expected. Since the medium used for the test was slightly alkaline

312

(with pH around 8), CAP would be expected to degrade into compound TP120, 21,

313

which was shown not to be toxic to D. magna. However, based on previous studies

314

on CAP stability,10, 15, 20 rapid transformation in water would be unlikely for CAP.

315

Moreover, the medium was renewed every three days and the actual concentrations

316

of CAP remained fairly constant. This proves that no considerable degradation of

317

CAP occurred during the incubation period. Another reason could be related to the

318

mode of action of CAP. CAP is a rapid-acting insecticide, yet under its mortality

319

threshold the daphnids survived and reproduced normally.

(11.6 µg/L), based on mean


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Comparing the acute and chronic EC50 values of CAP and other new age

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insecticides, it appears that CAP is one of the most toxic current-use insecticides to

322

D. magna. Generally, neonicotinoids, nicotinic acetylcholine receptor activators,

323

pose much lower risks to daphnids compared to CAP. CAP was also shown to be

324

the most toxic representative within its own group, the diamide insecticides. Higher

325

toxicities to daphnids are however, common for some representatives of the older

326

insecticide groups, which have a long history of widespread use, such as

327

carbamates, organophosphates and pyrethroids (Table 1). A large variation in acute

328

EC50 values for D. magna was observed within the carbamate and organophosphate

329

insecticides.

330

CAP and cyantraniliprole, another anthranilicdiamide insecticide exhibited similar

331

toxicities. For cyantraniliprole, EFSA35 reported a chronic EC50of 11.23 µg/L,

332

based on the mortality of adults and juveniles, which is three times higher than the

333

EC50 for CAP obtained in our study. No information about reproduction is provided

334

in the reports for cyantraniliprole and since in chronic tests usually effects on

335

reproduction are determined, it might be assumed that, just like for CAP,

336

cyantraniliprole only affected daphnid survival. Moreover, mortality at the highest

337

test concentrations (14.7 and 23.9 µg/L) occurred mostly between day 1 and day 3.

338

This toxicity pattern and its very potent action closely resemble those of CAP

339

(Figure 3). It seems that both insecticides activate the daphnid ryanodine receptors, 17 ACS Paragon Plus Environment


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340

resulting in an acute mortality above their respective threshold values. The different

341

modes of action of pesticides, which belong to the class of specifically acting

342

chemicals, lead to a large variation of ACRs.49The small ACR for CAP additionally

343

suggests that its toxic effect occurs immediately or not at all.This rapid lethal

344

effect, together with the normal development of the daphnids exposed to sublethal

345

CAP concentrations is reflected by the sharp acute and chronic concentration-

346

response curves.

347

The decrease in survival was also the only factor responsible for a decrease in

348

population growth rate, with an EC50 (3.5 µg/L) very similar to that for daphnid

349

immobility (3.7 µg/L). In agreement, EPA10 and EFSA14,

350

NOAEC value of CAP for D. magna to be 4.5 µg/L. This was based on mean,

351

measured concentrations and adult length, adult immobility after 21 days, total

352

living neonates after 21 days, and immobilized neonates after 21 days for neonates

353

exposed under unaerated, static-renewal conditions.

354

For the CAP transformation product TP1, no effect on immobilization was

355

recorded in the acute toxicity test, which coincides with the results reported by

356

EPA10 and EFSA.14, 15Additionally in our research also no effect on immobilization

357

and reproduction was observed in a 21-days chronic toxicity test. From the

358

chemical structure of CAP and TP1 (Figure 1) it can be seen that TP1 is formed by

359

the elimination of water from the CAP molecule which is followed by the 18 ACS Paragon Plus Environment

15

reported the chronic

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formation of a quinazolinone ring. This suggests that the transformation is causing

361

an inactivity of TP1 to daphnid ryanodine receptors. The chemical structure of the

362

phototransformation product TP2 strongly resembles that of TP1 (Figure 1). Based

363

on the structural similarity, it was expected that TP2 would also be less toxic than

364

CAP. Although some mortality occurred in the acute test at the two highest tested

365

concentrations, no significant effect on mortality and reproduction was observed in

366

the chronic test. From the literature,14, 15 only the data from a 24-h toxicity test with

367

TP2 tested up to 0.1 mg/L was known. No toxicity was observed in that study,

368

while our study demonstrated lack of toxicity also at even higher concentrations

369

(0.9 mg/L) upon chronic exposure.

370

Originally CAP was assigned to be persistent in soil and aquatic sediments.50

371

Therefore runoff from the soil could be a frequent source of CAP in surface water.

372

The labeling restrictions for products containing CAP are warning that the product

373

has a high potential for runoff for several months or more after it is applied on the

374

field. In a dissipation study on a rice field system,51 the half-life of CAP (CAP

375

formulations sprayed at 300 mL a.i. hm-2) was 16 days in soil (pH 6.2, OM content

376

2.52 %) and 0.85 days in water, with an initial CAP concentration of 28 µg/L.

377

Daphnid exposure to this concentration could lead to acute mortality, since in our

378

acute toxicity test 35% of the initial daphnids were immobilized after 24-h

379

exposure to 18.9 µg CAP /L and complete mortality of all exposed animals in the 19 ACS Paragon Plus Environment


Page 20 of 43

380

same test vessels followed the next day. Generally, in the aquatic environment CAP

381

was assigned to be moderately persistent52 and therefore chronic exposures of

382

aquatic organisms could also be expected. Additionally, with frequent runoffs,

383

aquatic organisms can also be exposed to pulse discharges of chemicals, which may

384

result in even more pronounced toxic effects.42

385

In natural waters with the pH above neutral, one of the degradation pathways

386

anticipated for CAP is spontaneous transformation to TP1. Unless exposed to the

387

prolonged high intensity irradiation,20 TP1 appears stable. However, despite the

388

possible chronic exposures, harmful effects on the daphnids are not expected based

389

on the results of the present study. The second degradation pathway of CAP

390

occurring in natural waters is induced by solar irradiation. Among the tree main

391

degradation products in this degradation pathway, TP2 was detected in the highest

392

concentrations in natural water and under the natural summer sunlight;53 TP2 is

393

harmless to the daphnids.

394 395

While generally the hazard of CAP to non-target organisms is low,10 the present

396

study demonstrated a high sensitivity of non-target micro-crustaceans to CAP.

397

However, the actual risk of CAP in water diminishes with its spontaneous or light-

398

induced degradation into two transformation products, showing no acute or chronic

399

toxic effects, based on the present study. 20 ACS Paragon Plus Environment

Page 21 of 43


400

401

Supporting Information Available: [1. Chronic survival-time relationships for

402

Daphnia magna exposed to TP1 and TP2. 2. Average measured concentrations of

403

chlorantraniliprole and TP2 in test solutions from the acute toxicity tests with

404

Daphnia magna. 3. Average measured concentrations of chlorantraniliprole and

405

TP2 in test solutions from the chronic toxicity test with Daphnia magna. 4.

406

Chemical analysis of actual test concentrations.] This material is available free of

407

charge via the Internet at http://pubs.acs.org.

408

409

410

ACKNOWLEDGEMENTS:

411

The authors wish to acknowledge and thank HenrikBarmentlo for the population

412

growth rate calculations, Chiara Cerli, Joke Westerveld and Peter Serne for

413

technical support, Hannah Härtwich for the help with chemical analysis and

414

FaizaKaiouh for supplying the daphnids. We also thank TanjaBleyenberg, Arne

415

Dits, Marian Schoorl and JeroenSchütt for sharing their experiences of toxicity

416

testing.

417

The authors gratefully acknowledge theSlovenianResearchAgency(ARRS) for the

418

financial support in the form of Research Program P1-0030. 21 ACS Paragon Plus Environment


Page 22 of 43

419

Table:

420

Table 1: Comparison of the acute (48-h) EC50 values of the toxicity of selected

421

insecticides for Daphnia magna Insecticide group 1 1 1 2 2 2 2 3 3 3 3 4 4 4 4 5 5 5

Insecticide chlorantraniliprole cyantraniliprole flubendiamide imidacloprid thiamethoxam thiacloprid clothianidin carbaryl pirimicarb methomyl aldicarb chlorpyrifos malathion dimethoate diazinon permethrin λ-cyhalothrin deltamethrin

Acute EC50 (µg/L) 9.4 11.6 20 > 60 84,000 > 100,000 > 85,100 100,800 - 119,000 0.016 16.0 28.7 583.0 0.6 28.1 1100 0.7-1.25 0.32 0.39 0.15

Test specification, notes 48-h 48-h 48-h 48-h 48-h 48-h 48-h 48-h 48-h 48-h 48-h 48-h* 48-h* 48-h 48-h 48-h 48-h 48-h** 48-h**

Reference Present study 10, 14, 15 35 36 37 38 39 40 41 42 43 44 44 45 42 46 47 48 48

422

Insecticide groups: 1 – diamide insecticides, 2 – neonicotinoids, 3 - carbamate

423

insecticides, 4 - organophosphate insecticides, 5 -pyrethroids

424

* Toxicity test performed with the formulated product. Filtered spring water was

425

used as a test media.

426

** Toxicity test started using 4-5 days old daphnids.

427


Page 23 of 43

428


Figures:

429

430

Figure 1: Chemical structures of chlorantraniliprole (CAP) (left) and its main

431

transformation products in water, transformation product 1 (TP1, middle, called

432

IN-EQW78 by the registrant) and transformation product 2 (TP2, right, called

433

IN-LBA23 by the registrant).

434



Page 24 of 43

435 436

Figure 2: Average mobility (% initial animals) of Daphnia magna (n=4) exposed

437

to chlorantraniliprole (µg/L) for 48-h (LC50 = 9.4 µg/L). The logistic curve

438

represents the fitted concentration-response relationship.

439

Error bars (in x and y) represent the standard deviation.

440


Page 25 of 43


441

442

Figure 3.Time-dependent (% control, left panel) concentration-dependent (%

443

control, right panel) mobility relationships for Daphnia magna exposed to

444

chlorantraniliprole (µg/L) for 21 days. On the right panel, the acute

445

concentration response curve is plotted beside the chronic one (LC50 = 3.7 µg/L),

446

indicating the corresponding 48-h LC50 value (9.4 µg/L).

447

Error bars on the right panel represent the standard deviation (n = 5 for 0.9 µg CAP/L, n = 6

448

for 3.0 µg CAP/L and n = 2 for 8.0, 9.5 and 10.8 µg CAP/L).

449



Page 26 of 43

450 451

Figure 4: Average per capita population growth rate (day-1) for Daphnia magna,

452

exposed to chlorantraniliprole (µg/L)for 21 days (EC50 = 3.5 µg/L).

453

Error bars represent the standard deviation (n = 5 for 0.9 µg CAP/L, n = 6 for 3.0 µg CAP/L

454

and n = 2 for 8.0, 9.5 and 10.8 µg CAP/L).

455


Page 27 of 43

456


TOC/Abstract art

457

458 459



460

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Daphnid Life Cycle Responses to the Insecticide Chlorantraniliprole

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