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Chemosphere 185 (2017) 1019e1029

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Lethal and sub-lethal effects of commonly used anti-sea liceformulations on non-target crab Metacarcinus edwardsii larvae

Paulina Gebauer a, *, Kurt Paschke b, d, Claudia Vera b, Jorge E. Toro c, Miguel Pardo c, d,Mauricio Urbina e

a Centro i~mar, Universidad de Los Lagos, Casilla 557 Puerto Montt, Chileb Instituto de Acuicultura, Universidad Austral de Chile, Casilla 1327, Puerto Montt, Chilec Instituto de Ciencias Marinas y Limnol�ogicas, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chiled Centro FONDAP de Investigaci�on en Din�amica de Ecosistemas Marinos de Altas Latitudes (IDEAL), Chilee Departamento de Zoología, Facultad de Ciencias Naturales y Oceanogr�aficas, Universidad de Concepci�on, Chile

h i g h l i g h t s

� Cypermethrin, deltamethrin, and azamethiphos affected 100% crab larvae at concentrations lower than used against sea-lice.� Hydrogen peroxide at the concentration used as an anti-sea lice treatment had lethal and sub-lethal effects on M. edwardsii zoea I.� Repeated exposure to azamethiphos (0.0625e0.5 mg L�1) increased mortality, but did not affect zoea I developmental time.� Chronic exposure to hydrogen peroxide (187.5e1500 mg L�1) had a lethal effect on larvae.

a r t i c l e i n f o

Article history:Received 8 February 2017Received in revised form10 July 2017Accepted 19 July 2017Available online 24 July 2017

Handling Editor: Jim Lazorchak

Keywords:CypermethrinDeltamethrinAzamethiphosHydrogen peroxideOrganophosphateCrustacean larvae

* Corresponding author.E-mail address: pgebauer@ulagos.cl (P. Gebauer).

http://dx.doi.org/10.1016/j.chemosphere.2017.07.1080045-6535/© 2017 Elsevier Ltd. All rights reserved.

a b s t r a c t

The pesticides used by the salmon industry to treat sea lice, are applied in situ via a bath solution and aresubsequently discharged into the surrounding medium. The effects of cypermethrin, deltamethrin,azamethiphos and hydrogen peroxide were assessed on the performance of Metacarcinus edwardsiilarvae, an important crab for Chilean fishery. All larvae were dead or dying after 30 min of exposure tocypermethrin and after 40 min to deltamethrin at concentrations 100 and 20 times lower (0.15 and0.1 mg L�1, respectively) than the concentrations and exposure times recommended by the manufacturers(CRM) to treat sea lice. Azamethiphos affected all larvae at a concentration 10 times lower than CRM.Hydrogen peroxide had the lowest detrimental effects, but at the CRM, 100% of the larvae were affected.Sub-lethal effects, i.e prolonged developmental time, were observed at concentrations lower than CRM.Repeated exposure to azamethiphos (0.0625e0.5 mg L�1) and hydrogen peroxide (188e1500 mg L�1) hadeffects on survival. In conclusion, the pesticides used against parasitic copepod tested here, negativelyaffect non-target crustacean larvae. Due to the product’s characteristics, the lethal effects of the pyre-throids probably are restricted to the time and area of application, while the action of azamethiphos mayextend to a wider area. Current data are insufficient to accurately dimension the effects of these com-pounds in the field. More research is required to evaluate the consequences of prolonged developmentaltimes and/or reduction in appendage mobility, so as the effects of these compounds on the pelagic andbenthic communities.

© 2017 Elsevier Ltd. All rights reserved.

1. Introduction

In the Southern Hemisphere, salmon and trout are intensivelyfarmed in the fjords and channels of Chilean Patagonia. Salmon

farming is susceptible to various bacterial, viral, and parasitic dis-eases, due to the high density of cultivation, requiring the use ofboth antibiotic and antiparasitic treatments (Burridge et al., 2010).Among them, sea lice pose a serious threat to productivity in bothsalmon and trout farming (Burridge et al., 2014). In global monetaryterms, costs associated to sea lice control equate to around 0.19 Vkg�1 of salmon produced (Costello, 2009). In Chile, the

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P. Gebauer et al. / Chemosphere 185 (2017) 1019e10291020

predominant parasite species is the copepod Caligus rogercresseyi.Control measures have included the application of chemical com-pounds (Aaen et al., 2015), such as organophosphates (since 1981)specifically azamethiphos (since 2013), synthetic pyrethroids (e.g.cypermethrin and deltamenthrin, used since 2007 (Aaen et al.,2015), and hydrogen peroxide to a lesser degree. Large volumesof these pesticides are discharged into the Chilean marineecosystem; for example, 677 kg of active compound cypermethrinand 197 kg of deltamethrin were discharged in 2012 (Helgesenet al., 2014). Despite these levels of discharge, very little is knownabout the potential negative effects on aquatic life.

These pesticides have a different mechanism of action. Organ-ophosphates are neurotoxics that inhibit the enzymatic activity ofacetylcholinesterase (AChE), the enzyme responsible for acetyl-choline hydrolysis (Intorre et al., 2004). Pyrethroids act on neuraltransmission by interfering with sodium channels (Miller andAdams, 1982), resulting in motor neuron depolarisation and re-petitive discharge in the nerve endings, whichmay lead to paralysisand death (Crane et al., 2011; Haya et al., 2005). Hydrogen peroxideacts as an oxidising agent generating gaseous oxygen, which re-mains trapped inside the cuticle of the crustaceans. These chemicalcompounds are applied in situ by enclosing net pens and bathingfish using a tarpaulin. After completion of the chemical treatment,the compound is then, released into the surrounding seawater.Dispersion and dilution rates of the chemicals depend on thecharacteristics of each pesticide and the hydrographic character-istics of the area. A residual chemical compound concentrationpersists in the area around the treated pens, which is likely toimpact non-target species. (Burridge et al., 2014; Ernst et al., 2014).Specifically, concentrations of cypermethrin between 0.185 and0.218 mgL�1 have been measured 25 m away from the net pentreated, 25min after final treatment (Hunter and Fraser,1995;Williset al., 2005), while concentrations of deltamethrin between 0.020and 0.040 mgL�1 have been detected 4e5 h after treatments withthis compounds (Ernst et al., 2014). For azamethiphos concentra-tions between 1 and 25 mg L-1 have being measured from 1 to1000 m away from the application area (Ernst et al., 2014).

Themajority of research assessing the lethal effects of pesticidesused on sea lice have been conducted on adult of non-targetcrustaceans, determining lethal and/or effective concentrations(LC50, EC50) (see more detailed Table 1 Ernst et al., 2001; Mayoret al., 2008; see more detailed Table 5 Van Geest et al., 2014).However, very little is known about the lethal effects of thesechemical compounds on the early life stages of decapod crusta-ceans. It is useful to note that LC50 has been calculated for Americanlobster (Homarus americanus) larvae for cypermethrin (Burridgeet al., 2000a, 2000b; Pahl and Opitz, 1999), deltamethrin(Burridge et al., 2014), azamethiphos (Burridge et al., 2000b, 2014;Pahl and Opitz, 1999), and hydrogen peroxide (Burridge et al.,2014), providing some indication of the expected effects.

Table 1Nominal concentrations of cypermethrin, deltamethrin, azamethiphos and hydrogen peconcentration recommended by the manufacturer. Exposure times for each compound a

Compounds Treatments-Concentrations

Control (seawater) A1 A2

Cypermethrin(mg L�1, 30 min)

0 0.15 0.5

Deltamethrin(mg L�1, 40 min)

0 0.1 0.25

Azamethiphos(mg L�1, 30 min)

0 1 3

Hydrogen peroxide(mg L�1, 20 min)

0 50 100

Sub-lethal effects, such as changes in locomotion, were reportedafter exposing adult American lobster to azamethiphos (Abgrallet al., 2000), and prawn (Palaemon serratus) to deltamethrin(�0.6 ng L�1) (Oliveira et al., 2012). In several species of free-livingcopepods, both swimming and feeding behaviours are altered byconcentrations of 5 mg L�1 hydrogen peroxide (Van Geest et al.,2014) and 0.098e0.32 mg L�1 cypermethrin (Barata et al., 2002).Damage by oxidative stress as protein carbonylation were reportedin H. americanus as a result of chronic exposure to 61 ng L�1 aza-methiphos, and lipid oxidation was reported in Penaeus monodonafter 4 days of exposure to 0.1 mg L�1 deltamethrin (Dorts et al.,2009). Both types of damage were observed in Procambarus clar-kii after exposure to 0.005 mg L�1 cypermethrin for 24 h (Burridgeet al., 2014). These previous studies indicate a range of both lethaland sub-lethal effects on non-target organisms from the applica-tion of the various families of pesticide chemicals.

Our aim was to assess the effects of commercial pesticides,commonly used in the treatment of C. rogercresseyi on fish farms, onthe larval performance of the South American crab speciesM. edwardsii. This commercially important crab species is abundanton the coasts of Southern Chile, accounts for nearly 80% of theartisanal crab fishery, and is an important component of the localeconomy (Chilean national fishery statistics, SERNAPESCA,2011e2014). The effects of acute (based on the manufacturers’recommendations, time and concentration) and chronic exposureto pesticides on the first larval stage (zoea I) of M. edwardsii wereinvestigated. Developmental time and survival were used asresponse variable; we also estimated the 50% lethal and effectiveconcentrations. Given the generality and broad action spectrum ofthe formulations used to treat sea lice in Chilean salmon-farming,we hypothesize cypermethrin, deltamethrin, azamethiphos andhydrogen peroxide will negatively affect the developmental timeand survival of the zoea I of M. edwardsii.

2. Materials and methods

2.1. Larvae collection

Ten ovigerousM. edwardsii females were kept in containers withcirculating seawater until larvae hatched (~12 �C, 32 PSU for 1month). Recently hatched zoea I were collected and activelyswimming larvae were selected for the exposure to cypermethrin(Betamax®), deltamethrin (Alpha Max®), azamethiphos (Calfree®),and hydrogen peroxide (Hyperox®).

2.2. Acute exposures

Four independent experiments were carried out, in whichdifferent groups of zoea I larvae (2800 larvae per experiment) wereexposed to each of the four compounds. For each compound

roxide, and exposure times for the acute experiments in M. edwardsii larvae. CRM:re the recommended by the manufacturers for treatment of C. rogercreseeyi.

A3 A4 A5 (CRM) A6 A7

1.5 5 15 30 60

0.5 1 2 3 8

10 30 100 300 500

300 750 1500 2000 3000

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(experiment), a working solution was prepared in UV-sterilisedseawater and filtered using a 1 mm filter. The required compoundsolution volume was extracted by repetitive pipette and added tothe glass exposure containers filled with 3 L filtered and sterilisedseawater, mixed for 2 min with a glass rod, in order to reach thecorresponding concentrations before larvae were exposed. In eachexperiment, different larvae groups were exposed to one of sevendifferent concentrations (7 replicates per concentration,comprising 50 larvae each; 350 larvae per concentration) of each ofthe four compounds, plus a control group. The exposure times werebased on the time recommended by manufacturer for treatmentagainst C. rogercreseeyi (Table 1); the control group experienced thesame handling as other treatments but it was only exposed toseawater.

Following exposure, larvae were transferred to clean containersof filtered seawater and rinsed three times (one minute each time).Immediately after rinsing, and before being placed into the follow-up containers, the larvae were observed and registered into one offour groups: a) dead; b) dying A, those lying on the bottom of thecontainer with weak appendage movements only observable understereoscopic microscope; c) dying B, those on the bottom of thecontainer, unable to swim but with active appendage movement,observable without stereoscopic microscope; and d) alive (i.e.actively swimming larvae).

Replicates were kept in 800 mL follow-up containers filled withseawater, filtered at 1 mm, without aeration, at 15 �C, 32 PSU and12:12 light:darkness photoperiod. Daily seawater and food (freshlyhatched Artemia sp, nauplii) were changed. Larvae were countedand classified as alive, dying (dying A or B), or dead, and thepresence of exuviaewas checked. The experiments weremonitoreduntil all larvae were classified as either dead or moulted to larvalstage zoea II.

2.3. Chronic exposures

Chronic exposure experiments were only carried out for aza-methiphos and hydrogen peroxide, since 100% of the larvaeexposed to the pyrethroids (i.e. cypermethrin and deltamethrin),were either dead or dying 24 h post-exposure at the lowest con-centrations (0.15 and 0.1 mg L�1, respectively). Each experimentconsisted of 1750 larvae divided into four concentrations (Table 2),and a control group consisting of clean seawater. Each treatmentand control group contained seven replicates with 50 zoea I larvaeper replicate.

The rearing conditions (i.e. water changes, feeding, selection,and larvae classification) remained the same as for acute exposure.Larvae were exposed daily over a seven-day period at the con-centrations and exposure times indicated in Table 2. This chronicexposure period is close to the moulting time of zoea I to zoea II, sothe exposure of only zoea I stage was assured. Each replicate groupwas exposed independently over the full period of the experiment.

Table 2Nominal concentrations of azamethiphos and hydrogen peroxide, and exposuretimes for the chronic experiments in M. edwardsii larvae. CRM: concentration rec-ommended by the manufacturer.

Compound Treatments-Concentrations

Control (seawater) C1 C2 C3 C4

Azamethiphos(mg L�1, 30 min)

0 0.0625 0.125 0.25 0.5

Hydrogen peroxide(mg L�1, 20 min)

0 188 375 750 1500

2.4. Statistical analysis

The effect of each chemical compound on the developmentaltime and mortality of the first larval stage (zoea I) was assessedusing analysis of deviance, considering a gamma distribution forthe developmental time and a binomial distribution for the mor-tality. Pairwise comparison were carried out using Tukey test,implemented in the R library multicomp (Bretz et al., 2016). Thehomogeneity of variance was assessed using Levene’s test. The 50%lethal and effective concentrations for the chemical compoundswere also calculated, adjusting the survival data to a sigmoidaldose-response curve to calculate the concentration at which 50% ofthe larvae died (LC50) or were died or dying (EC50). The analyseswere carried out using R, version 3.2.4.

3. Results

3.1. Acute exposure

3.1.1. CypermethrinLarvae exposed to cypermethrin for 30 min presented 100%

mortality at 0.5, 1.5, 5, 15 (CRM), 30 and 60 mg L�1. At 0.15 mg L�1, alllarvae were classified as dying A, and 78.09± 1.53% of these diedwithin 24 h after exposure while the rest remained dying. After48 h, accumulated mortality for this treatment was 98.83± 2.90%(Fig. 1) reaching 100% mortality at 96 h post-exposure. Controlmortality was 21.48± 3.78% after 24 h, andwere the only larvae thatsurvived (34.44± 3.03%) and moulted to zoea II (7.77 ± 0.11 days).

3.1.2. DeltamethrinAfter 40 min of exposure to 0.1, 0.25, 0.5 and 1 mg L�1 delta-

methrin, 100% of M. edwardsii larvae were classified as dying B(unable to swim but with observable active appendagemovement).At the three highest concentrations (2, 3, 8 mg L�1), ~33% of thelarvae died, and the remaining presented only weak appendagemovements (dying A). All larvae in the control swam actively. Therewere significant differences in mortalities between groups 24 hpost-pesticide exposure (c2(48) ¼ 50.68; P < 0.0001), and pairwisecomparisons (Tukey) revealed four groups: a) control (3.50± 0.96%mortality), b) the treatments 0.1 and 0.25 mg L�1 (between9.70± 1.58% and 11.10± 1.68% mortality), c) treatments 0.5 and1 mg L�1 (~50% mortality), and d) the three highest concentrations(between 89.80± 1.64% and 91.66± 1.51% mortality). The LC50calculated at 24 h was 1.252 ± 0.046 mg L�1, roughly 37% lower thanthe concentration recommended by manufacturer (2 mg L�1).However, it is worth noting that, while the LC50 is an estimate basedon mortality, 100% of the larvae were negatively affected by thisconcentration. Over the same period, dead and dying larvae werefound in all treatment groups, and the accumulated mortality was100% in all concentrations 48 h post-exposure (Fig. 2). The larvaefrom the control group were the only survivors (35.09± 3.37%), andmoulted to next stages at 8.37 ± 0.14 days.

3.1.3. AzamethiphosThe larvae in the control, 1 and 3 mg L�1 azamethiphos treat-

ments swam actively post-exposure, while the larvae exposed to10, 30, 100, 300, and 500 mg L�1 azamethiphos were all dying after30 min of exposure. In the 10 and 30 mg L�1 treatments, larvae werein category dying B, moving actively the appendages butcompletely unable to swim or capture food. Larvae exposed at100 mg L�1 presented weak appendage movement and curledtelson, and those exposed to 300 and 500 mg L�1 concentrationswere classified as dying A. At 24 h post-exposure, there was sig-nificant larval mortality among the treatments (c2(48) ¼ 49.77;P < 0.0001). Tukey pairwise comparisons identified two groups: a)

Fig. 1. Cumulative percent of dead and dying larvae 24 and 48 h following 30 min exposure to cypermethrin at different concentrations. Arrow indicates the concentrationrecommended by the manufacturer (CRM). Data represented at means ± SE.

Fig. 2. Cumulative percent of dead and dying larvae 24 and 48 h following 40 min exposure to different concentrations of deltamethrin. Arrow indicates concentration recom-mended by the manufacturer (CRM). Data represent means ± SE.

P. Gebauer et al. / Chemosphere 185 (2017) 1019e10291022

P. Gebauer et al. / Chemosphere 185 (2017) 1019e1029 1023

0, 1, and 3 mg L�1, with a mean mortality of 39.13% and b) the othertreatments (from 10 to 500 mg L�1), with a mortality that variedbetween 91 and 100%. The LC50 at 24 h post-exposure was2.84 ± 0.39 mg L�1. The effect of the two lowest azamethiphosconcentrations (1 and 3 mg L�1) 24 h post-exposure can be clearlyseen in the number of dead and dying larvae. These treatmentsshowed 60% of larvae dead and dying versus 34% in the controlgroup (c2(48)¼ 42.18; P < 0.0001). The EC50 calculated for 24 h post-exposure (dead þ dying) was 0.94 ± 0.15 mg L�1.

At 48 h post-exposure, mortality in the 1 and 3 mg L�1 treat-ments increased to 30e58%, reaching roughly 80% accumulatedmortality compared to only 38% in the control group (Fig. 3A). In thecontrol group, 27.89± 1.73% of the larvae survived, moulting to zoeaII after 7.34 ± 0.05 days. In treatments 1 and 3 mg L�1, only 1.71% and3.04% of the larvae reached zoea II after 7.4 ± 0.24 and 7.11 ± 0.11days, respectively (Fig. 3B). Significant differences were observed inthe total zoea I mortality between treatment and control groups(c2(18) ¼ 26.95; P < 0.0001), while no difference was shown indevelopmental time (F(2-11) ¼ 0.917; P > 0.05).

3.1.4. Hydrogen peroxideAfter 40 min of exposure, 100% larvae at 50, 100 and 300 mg L�1

hydrogen peroxide and control swam actively, while 85% of thelarvae exposed to 750mg L�1 swam actively and the remaining 15%,lay on the bottom but still presenting active movement of theirappendages. The larvae exposed to 1500, 2000, 3000 mg L�1 wereclassified as dying B. The percentage of affected larvae(dead þ dying) 24 h post-exposure, for concentrations between0 and 750 mg L�1, ranged between 16.27± 1.89% and 22.71± 2.05%.The 1500 mg L�1 treatment recorded 41.43± 2.50% of larvaeaffected, with 23% dying. The 2000 and 3000 mg L�1 treatmentsaffected more than 50% of the larvae. At 48 h post-exposure, 39.2%of larvae in the 1500 mg L�1 treatment group (CRM) were nega-tively affected, whilst 69.80± 2.30% and 98.90± 0.50% of larvae inthe 2000 and 3000 mg L�1 treatments were affected, respectively(Fig. 4A). The EC50 (dead þ dying) calculated 48 h post-exposurewas 1269.61 ± 41.33 mg L�1. Comparing larval mortality 72 h and96 h post-application showed few variations. The accumulatedmortality presented significant differences between treatments(c2(48) ¼ 150.99; P < 0.001): concentrations up to 750 mg L�1showed no differences (Tukey test P > 0.05), with a mean mortalityof 30.2± 2.07%. The accumulated mortality of the other treatments,ranged between 41.9± 2.5% and 98.3± 0.6%. The EC50 (deadþ dying)after 72 h was 1130.19 ± 42.14 mg L�1 hydrogen peroxide, andslightly lower after 96 h (1036.25 ± 43.44 mg L�1).

Both zoea I developmental time and survival were affected byexposure to the different concentrations of hydrogen peroxide (F(6-41)¼ 319.06; c2(48)¼ 107.16; P< 0.0001, Fig. 4B). The developmentaltime of the control larvae was 8.54 ± 0.08 days, and those of the 50,100, and 300 mg L�1 treatments were 8.65 ± 0.09, 8.2 ± 0.08, and8.63 ± 0.08 days, respectively (Tukey test P > 0.05). Larvae exposedto 750, 1500, 2000, and 3000 mg L�1 hydrogen peroxide suffereddelayed development, moulting to zoea II after 11.58 ± 0.11,11.78 ± 0.12, 12.13 ± 0.13, and 12 days, respectively, these concen-trations showed significant differences with the control and 50,100and 300 mg L�1 treatments (Tukey test, P < 0.05). There were sig-nificant differences in survival between treatments (c2(48)¼ 107.16;P < 0.0001), and pairwise comparisons (Tukey) revealed threegroups: a) 0, 50, 100, and 300 mg L�1 treatments (55.89± 2.01%survival), b) 750 and 1500 mg L�1 treatments, roughly 35% survival,and c) 2000 and 3000 mg L�1 treatments (11.4 ± 0.94 and 0.28%,respectively).

3.2. Chronic exposure

3.2.1. AzamethiphosMortality of M. edwardsii larvae 24 h after first exposure to

azamethiphos showed significant differences between the controland treatment groups (c2(30) ¼ 121.97; P < 0.001). The greatestincrease in mortality for all concentrations occurred 48 h post-exposure, after the second exposure to azamethiphos (Fig. 5A).Differences in survival between control and the treatment groupsremained throughout the experiment, with the exception that day-seven survival in the 0.5 mg L�1 treatment differed from the othertreatments, which coincided with the first day of moults to zoea II.The developmental time of zoea I in the control group was9.51 ± 0.07 days; this presented no difference with the treatments(F(4-30) ¼ 1.31; P > 0.05), which ranged between 9.37 ± 0.23 and9.87 ± 0.11 days, at 0.5 and 0.00625 mg L�1, respectively (Fig. 5B).The survival of the first larval stage was affected significantly(c2(30) ¼ 104.56; P < 0.0001). The control had the highest survival(78.28± 2.22%), followed by the 0.0625, 0.125, and 0.25 mg L�1

treatments, with survival roughly 57%; the percent survival of the0.5 mg L�1 treatment was 45.71 ± 2.66.10%. (Fig. 5B).

3.2.2. Hydrogen peroxideBetween 16.28 ± 1.97 and 27.42± 2.39% of larvae were affected

(dead þ dying) (Fig. 6), significantly different from the control(c2(30) ¼ 51.46; P < 0.001) 24 h after the first exposure to hydrogenperoxide. Following two exposure at 1500 mg L�1, over 50% of thelarvae were affected, while at lower concentrations(187.5e750 mg L�1), between 22.2% and 35.1%. After the thirdhydrogen peroxide exposure of 375 and 750 mg L�1, more than 60%of the larvae were affected. Four groups were distinguished sta-tistically (Tukey test) at 72 h: a) the control, with 15.5% of larvaeaffected, b) the 187.5 mg L�1 treatment, with 24.9%, c) the 375 and750 mg L�1 treatments, with roughly 62%, and d) the 1500 mg L�1

treatment, with 88.6% larvae affected (Fig. 6A). Close to 100% of thelarvae were affected after the fourth exposure to 1500 mg L�1 andthe four groups described above were maintained. No larvae wereactive after 6 exposures at 375 and 750 mg L�1. After 7 exposure,1.43% of larvae (zoea I) in the 187.5 mg L�1 treatment moulted tozoea II (9.22 ± 0.33 days). The rest of the larvae remained as zoea Ifor a maximum of 22 days. The control larvae moulted to zoea II onaverage after 9.51 ± 0.22 and showed 78.28± 2.2% survival (Fig. 6B).

4. Discussion

Zoea I larvae of M. edwardsii, are negatively impacted by all thecommonly used pesticides for the copepod parasite C. rogercresseyi(deltamethrin, cypermethrin, azamethiphos and hydrogenperoxide) tested here. M. edwardsii is of crucial socioeconomicimportance in southern Chile, and as its distribution coincides withthe salmon farming industry, it will likely be frequently exposed tothese pesticides. The concentrations and exposure times recom-mended by the manufacturers have lethal effects on the zoea Iunder acute exposure, with cypermethrin, deltamethrin, and aza-methiphos producing 100% mortality. Acute exposure to hydrogenperoxide at 1500 mg L�1 (CRM) generates both lethal (~67% mor-tality) and sub-lethal effects (delayed moult to zoea II in 38% of thesurviving larvae). Chronic exposure to hydrogen peroxide at con-centrations between 187.5 and 1500 mg L�1 provokes high mor-tality, but has no effect on the developmental time of the fewsurviving larvae.

Chronic exposure of M. edwardsii zoea I for 7 days to azame-thiphos at the concentrations used in this study have no effects ondevelopmental time, but do increase mortality by approximately20%. Exposure to 0.5 mg L�1 for 30 min daily for 7 days led to a ~50%

Fig. 3. A) Cumulative percent of dead and dying larvae 24 and 48 h following 30 min exposure to different concentrations of azamethiphos. B) Developmental time (days, bars) andsurvival of the first larval stage (zoea I) (triangle). Arrow indicates concentrate recommended by the manufacturer (CRM). Data represent means ± SE.

P. Gebauer et al. / Chemosphere 185 (2017) 1019e10291024

mortality; the same percentage was recorded in the acute experi-ments at 1 mg L�1 after 24 h post-exposure.

The two pyrethroids, cypermethrin and deltamethrin, wereextremely toxic for M. edwardsii larvae, as has been described forother crustaceans. In the case of cypermethrin, 100% of the

M. edwardsii larvae died in less than 24 h post-exposure at a con-centration 10 times lower (1.5 mg L�1) than the manufacturer’srecommendation (15 mg L�1), and 48 h post-exposure at a con-centration 100 times lower (0.15 mg L�1). This chemical compoundwas lethal and acted quickly against M. edwardsii larvae. Its high

Fig. 4. A) Cumulative percent of dead and dying larvae 24, 48, 72, and 96 h following 20 min exposure to different concentrations of hydrogen peroxide. B) Developmental time(days; bars) and survival of the first larval stage (zoea I) (filled triangle). Arrow indicates concentration recommended by the manufacturer (CRM). Data represent mean ± SE.

P. Gebauer et al. / Chemosphere 185 (2017) 1019e1029 1025

toxicity has been reported in larvae of Homarus americanus(Burridge et al., 2000a, 2000b; Pahl and Opitz, 1999), with a LC50varying between 0.06 and 0.66 mg L�1 depending on exposure time(48 h and 5min, respectively) and larval stage. In addition, a LC50 of0.17 mg L�1 after 4 h exposure has been reported in larval stages offresh-water copepods (Wendt-Rasch et al., 2003). The effect ofdeltamethrin has been assessed in American lobster larvae

(Burridge et al., 2014), with an estimated LC50 ranging from 0.0006to 0.0017 mg L�1 after 24 h exposure, and an estimated LC50 of0.0035 mg L�1 after one hour exposure. In the case of M. edwardsiilarvae, exposure to a concentration of 0.1 mg L�1 for only 40 mincaused 100% mortality after 48 h of exposure. Although difficult tocompare the sensitivity of different species, due to differences intheir mass and exposure conditions, the available information

Fig. 5. A) Percentage of dead larvae after 30 min daily exposure to different concentrations of azamethiphos, at 24, 48, 72, and 96 h. B) Developmental time (days; bars) and survivalof the first larval stage (zoea I) (filled triangle) after daily exposure to different concentrations of azamethiphos over 7 days. Data represent means ± SE.

P. Gebauer et al. / Chemosphere 185 (2017) 1019e10291026

Fig. 6. A) Percentage of larvae affected (dead þ dying) after 20 min daily exposure to different concentrations of hydrogen peroxide, at time points 24, 48, 72, and 96 h. B)Developmental time (days; bars) and survival of the first larval stage (zoea I) (filled triangle) after daily exposure to different concentrations of hydrogen peroxide over a seven-dayperiod. Data represent means ± SE.

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P. Gebauer et al. / Chemosphere 185 (2017) 1019e10291028

indicates that concentrations below manufacturer recommenda-tions produce lethal effects in the larval stages of the crustaceanspecies assessed.

For azamethiphos, a similar LC50 (1e3 mg L�1) was obtained forM. edwardsii zoea I as for larval stages I, II, III and IV of Homarusamericanus (Burridge et al., 1999); however the exposure periodwas much longer in the American lobster (48 h vs. 30 min). Thisindicates a greater sensitivity in M. edwadsii larvae, which may bedue to differences in the larvae mass (dry weight, M. edwardsii:15 mg vs. H. americanus: 899 mg, Pandian, 1970). As with cyper-methrin and deltamethrin, negative effects are found well belowthe concentration recommended by the manufacturer (100 mg L�1).

The effect of hydrogen peroxide on crustacean larval stages hasalso been studied in H. americanus larvae; with the LC50 24 h post-exposure for stage I larvae being calculated as 1637mg L�1 with onehour’s exposure (Burridge et al., 2014). This value is very similar tothat for M. edwardsii zoea I (1642 mg L�1) with 20 min exposure.Although the LC50 found is higher than the recommended1500 mg L�1 used to treat C. rogercresseyi, immediately followinghydrogen peroxide exposure at this concentration, 100% of theM. edwardsii larvae were unable to swim or feed. If this conditionwere to occur in the field, the affected larvae would almostcertainly die, because they would either precipitate, be carriedaway by the currents, or become vulnerable to predators. The lethaleffects reported in the present study may therefore underestimatethe effects of hydrogen peroxide on non-target species under nat-ural conditions.

In Chile, bathing with pyrethroids (cypermethrin and delta-methrin) and the organophosphate azamethiphos is coordinatedthrough a National Fisheries and Aquaculture Service (SERNA-PESCA) application timetable, normally consisting of roughly 8 daysof pyrethroids or azamethiphos application and then 8 dayswithout application. Under this regime, non-target organisms maysuffer prolonged exposure to chemical compounds. Chronic expo-sure of M. edwardsii larvae to azamethiphos concentrations�0.5 mg L�1 provoked a 24% increase in mortality. Thus, parasitecontrol may cause greater harm to non-target species than has beenpreviously thought. Furthermore, several net pens may be treatedin a relatively small area, increasing the potential for repeated orchronic exposure. A large part of Chilean salmon-farming occurs infjords, which often containmore than one salmon farm. If thewaterexchange rate is low, these zones could be exposed to both repeatedand chronic effects. Repeated exposure to azamethiphos for shortperiods causes additional negative effects in the American lobster(Burridge et al., 2000a), and the possibility of cumulative effects innon-target species resulting from different treatments cannot bediscounted in these environments.

Pyrethroids have low solubility in water (Burridge et al., 2010;Zhou et al., 1995) and adhere to particulate matter, making themless available in the water column and more likely to accumulate inthe sediment (Mayor et al., 2008). Although pyrethroids are highlytoxic pesticides, their direct effects in the aqueous phase areprobably manifested in a restricted time and space around thetreated net pens. Due to their high affinity with organic matter,pyrethroids would represent a greater risk for the benthic com-munity and organisms that ingest particulate matter. On the otherhand, azamethiphos has low absorption and low Kow, meaning thatit persists for longer time in the water column than pyrethroids(Ernst et al., 2014). These azamethiphos characteristics indicate thatits aqueous toxicity presents a higher risk, and sediment accumu-lation is a less important route for its disappearance from the watercolumn (Ernst et al., 2014).

Rapid cypermethrin dispersion and dilution occur after releasefrom salmon pens, and a single application of the product mayresult in a plume of up to 1 km2 (Ernst et al., 2001; Hunter and

Fraser, 1995). In a study carried out in Scotland, the highest con-centrations of cypermethrin (0.187 mg L�1) were measured 25 m(downstream) from the treated net pen, 25 min after application(Hunter and Fraser, 1995), Willis et al. (2005) estimate similarvalues for cypermethrin 25 min after final treatment (0.218, 0.197and 0.195 mg L�1). Furthermore, the cypermethrin concentrationremains above 0.074 mg L�1 up to 30 min after release (Hunter andFraser, 1995), and cypermethrin levels around pens 4e5 h afterapplication were measured between 0.020 and 0.040 mg L�1 (Ernstet al., 2001). Concentrations between 0.180 and 0.018 mg L�1 ofdeltamethrin have been measured 1 and 100 m, respectively fromtheir application point (Ernst et al., 2014), and azamethiphos con-centrations of approximately 25 and 1 mg L�1 were measured 1 and1000 m, respectively, from their application point. These dataindicate that cypermethrin concentrations, which this studydemonstrated to be lethal to M. edwardsii larvae (0.15 mg L�1), canpersist in the plume for at least 25e30 min. Deltamethrin con-centrations �0.1 mg L�1 may be present in the immediate sur-roundings of the net pen where the product has been applied,indicating that the direct effect of these pyrethroids is restricted inapplication time and area. For azamethiphos, the distance of actioncould extend up to 1000 m, presenting concentrations close to theestimated EC50 (0.94 ± 0.15 mg L�1) at that distance.

The direct lethal effects of pyrethroids on crustacean larvae areprobably restricted in time and space to the application point, butthe action of the organophosphate azamethiphos may affect alarger area of the water column. However, there is insufficient in-formation to dimension the real effects of the repeated applicationof these compounds over time and their potential synergistic ef-fects with other products. Further studies are needed on the effectsof these chemicals on the meroplankton, and on their potentialaccumulation in sediments, in order to examine broader implica-tions for both pelagic and benthic communities.

In conclusion, chemical compounds used to treat C. rogercressegiectoparasite have detrimental effects on the survival and devel-opmental time of M. edwardsii larvae, and potentially on manyother non-target species. This is due to un-specificity and broadaction spectrum of the formulations used to treat sea lice. Furtherresearch is needed on the pesticide impacts on non-target speciesto assess the broader effects of these compounds on marine eco-systems and move towards a sustainable and competitive aqua-culture industry.

Compliance with ethical standards

The authors declare no conflict of interest. All applicable inter-national, national, and/or institutional guidelines for the care anduse of animals were followed.

Acknowledgements

This work was supported by FIPA 2014e65. We are grateful toIgnacio Retamal for technical assistance with the rearing experi-ments. The authors would like to thank Dr.(c) KarenMiddlemiss forreviewing the language of this manuscript.

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Lethal and sub-lethal effects of commonly used anti-sea lice formulations on non-target crab Metacarcinus edwardsii larvae1. Introduction2. Materials and methods2.1. Larvae collection2.2. Acute exposures2.3. Chronic exposures2.4. Statistical analysis

3. Results3.1. Acute exposure3.1.1. Cypermethrin3.1.2. Deltamethrin3.1.3. Azamethiphos3.1.4. Hydrogen peroxide

3.2. Chronic exposure3.2.1. Azamethiphos3.2.2. Hydrogen peroxide

4. DiscussionCompliance with ethical standardsAcknowledgementsReferences