Aquatic Toxicology 163 (2015) 102108

Contents lists available at ScienceDirect

Aquatic Toxicology

j o ur na l ho me pag e: www.elsev ier .com/ locate /aquatox

Water inain Salm

Pamela O as Viviana Ca Laboratorio F en ProNaturales, Univb Departamentc Plataforma ded Laboratorio de Laboratorio d cultadde Chile, Valdiv

a r t i c l e i n f o

Article history:Received 4 FebReceived in reAccepted 28 MAvailable onlin

Keywords:DidymoSperm cellsToxity

a b s t r a c t

1. Introdu

Didymosdiatom. Thfound in thehas been coernment ofInternationvironment,and perturbwater syste

CorresponCatlica de Te

E-mail add

http://dx.doi.o0166-445X/ ruary 2015vised form 26 March 2015arch 2015e 8 April 2015

Didimosphenia geminata (didymo), has become a powerful and devastating river plague in Chile. Asystem was developed in D. geminata channels with the purpose evaluating the effects of water pollutedwith didymo on the activation of Atlantic salmon (Salmo salar) spermatozoa. Results indicate that semen,when activated with uncontaminated river water had an average time of 60 21 s. When using Powermilt,(a commercial activator), times of 240 21 s are achieved, while rivers contaminated with D. geminataachieve a motility time of 30 12 s. Interestingly enough, the kinetic parameters of VSL, VCL and VAPshowed no signicant changes under all of the conditions. Furthermore, the presence of D. geminatareduces activation time of the samples as the cells age, indicating increased effects in spermatozoa thatare conserved for more than 5 days. D. geminata has antioxidant content, represented by polyphenols;200 ppm of polyphenol were obtained in this study per 10 g of microalgae. Spermatozoa exposed to theseextracts showed a reduction in mobility time in a dose dependent manner, showing an IC50 of 15 ppm. Theresults suggest an effect on spermatozoa activation, possibly due to the release of polyphenols present incontaminated rivers, facilitating the alteration of sperm motility times, without affecting the viability orkinetics of the cells. These ndings have important implications for current policy regarding the controlof the algae. Current control measures focus on the number of visible species, and not on the compoundsthat they release, which this study shows, also have a problematic effect on salmon production.

2015 Elsevier B.V. All rights reserved.

ction

phenia geminata (D. geminata) is a unicellular benthicis microalgae known as didymo in Chile, has been

river waters of southern Chile (Riveraet al., 2013), andnsidered a plague in freshwater sources by the Gov-

Chile and can affect sh population (Reid et al., 2012).al studies indicate that D. geminata alters the microen-

reduces the sh population (Clearwater et al., 2010)s aquatic macro-vertebrate communities and drinkingm lters (Bergey et al., 2010; Gillis and Chalifour, 2010;

ding author at: Av. Las Mariposas S/N, Campus Dr. Rivas, Universidadmuco, Temuco, Chile. Tel.: +56 45205564.ress: jparodi@uct.cl (J. Parodi).

Kilroy et al., 2009). Recently, a toxic effect of microalgae on com-munities in contaminated rivers was described (Larned and Kilroy,2014). The removal of the microalgae, also introduces damagesto rivers (Larned and Kilroy, 2014). In Chile, there are no stud-ies that conrm this hypothesis and we must control these effectswith laboratory research to support eld observations (Rivera et al.,2013). Fish spermatozoa are immobile in the ejaculate, and it isonly after suffering osmotic shock in water that they are activatedand begin to move or swim (Alavi et al., 2009; Takei et al., 2012;Vladic and Jarvi, 2001). Spermatozoa are labile cells and are affectby aquatic contamination (Hatef et al., 2013). In the laboratory,one can replicate this effect using water from rivers or commer-cial solutions (Figueroa et al., 2013). Different kinetic parameterscan be observed, including mobility time of the masses, either man-ually (Ubilla and Valdebenito, 2011) or by computerized systems(Hu et al., 2013). This is a way of measuring their cellular function

rg/10.1016/j.aquatox.2015.03.0222015 Elsevier B.V. All rights reserved.contaminated with Didymosphenia gemo salar spermatozoa activation times

livaresa, Paola Orellanaa, Guillermo Guerraa, Mathavezd, Alfredo Ramireze, Jorge Parodia,

isiologa de la Reproduccin, Escuela de Medicina Veterinaria, Ncleo de Investigacin ersidad Catlica de Temuco, Chileo de Ingeniera en Obras Civiles, Universidad de Santiago de Chile, Chile

Investigacin en Ecohidrologa y Ecohidrulica, EcoHyd Ltda, Chilee Investigacin y Educacin, Tonalli Ltda, Chilee Criobiologa y Anlisis de Funcionalidad Espermtica. Instituto de Ciencia Animal, Faia, Chileta generates changes

Peredo-Paradab,c,

duccin Alimentaria, Facultad de Recursos

de Ciencias Veterinarias, Universidad Austral

P. Olivares et al. / Aquatic Toxicology 163 (2015) 102108 103

and the effects of molecules within their function, as motility andviability are related (Parodi, 2014) and computer assisted semenanalysis (CASA) can be used for evaluated toxic effect on spermato-zoa (Kime et al., 1996). Microalgae belonging to the family of browndiatom algapolyphenolInterestingldants and pon cancer cthat a certaibered that effect of a m

We evalAtlantic salmmicroalgae The same teand with Pofrom the dithe effects ostudied.

2. Method

2.1. Collect

D. geminin winter athe laboratand substracollected.

2.2. Protoco

Collectewater systeratory. The the articiadistilled waumn of watmaintainedtemperaturing gel syst71009 Plasetures waterto a 1 diamfor water dscopic and mwere record

2.3. Mortal

The morwith clear Techno Co.absence of as granularGeminata cpercentageviable formimages, a Nwas used foison with tFor this, thefor 10 min, viability of

observed, it is indicated as a viable form of D. geminata (Jellymanet al., 2010).

2.4. Extraction of polyphenols and confocal images

emintly g

zed wwithin inple

d anh a ded bto a med; fsity rozeotocowas ache

440 nyellol cohe sa, obt

differageJ

llect

electcomi

of Tetizee orainters

opor CaClgSOed

ctinged onicklyhe sainatwas kenntil

nalyz in tlocith thvarioa 10copesiblexim

atisti

resu An Aatione are rich in some molecules, such as antioxidants ands such as diadinoxhantina (Lohr and Wilhelm, 1999).y, several researches describe the benets of antioxi-olyphenols in cellular models. However, its toxic effectsells (Korkina et al., 2009) are also described, indicatingn dose may be lethal to cell groups. It should be remem-the dosage perfectly denes the positive or negativeolecule in a cell model.uated the effect of D. geminata in the activation ofon sperm. We used water that was contaminated with

to evaluate motility time, kinetics and cell viability.sts were carried out with uncontaminated river waterwermilt, a commercial activator. Polyphenols extracteddymo were used to evaluate a possible mechanism ofbserved from D. geminata on the spermatozoa functions

s

ion of D. geminata samples

ata was collected in the Futaleufu and Biobo riversnd spring of 2014. The samples were transported toory in plastic, closed dark boxes, at 10 C. River waterte (river rocks) colonized by the microalgae were also

l for maintenance of the D. geminata samples

d samples were kept in aquariums, closed recirculatingms called articial rivers, for observation in the labo-rocks contaminated with D. geminata were arranged inl rivers, adding 50% of the original river water, plus 50%ter, (total volume of 14 l) making sure to leave a col-er of about 15 cm over the rocks. Articial rivers were

with an expanded polystyrene insulate cover and thee was reduced to an average of 12 C through a cool-em. The water was kept in constant ux, using a modelt-Italy brand motor with 30 W potency. This device cap-

from the nal part of the articial river, and is connectedeter PVC pipe which returns to the origin of the river

ischarge, achieving constant ow and aeration. Micro-acroscopic changes in the aquarium with D. geminata

ed daily.

ity studies of D. geminata

tality of D. geminata was observed by visual inspectioneld microscopy, using an inverted Meijie (VT series,, Ltd., Japan) microscope, observing the presence orgranules within the cytoplasm, denominating them

forms (Root and OReilly, 2013). The numbers of D.ells, through a 40 lens, were counted, recording the

of those containing granules within the cytoplasm as a of indication. In order to improve the documentation inomarsky microscope at an objective of 40 (Olympus)r observation of intracellular structures and compar-he viability obtained using modied neutral red dye.

samples were left in a 0.01% neutral red dye solutionwhich has been reported as a means of assessing the

the D. geminata. When a granular red coloration is

D. gsequendeionisound in 1 mthe samagitatethrougdescribjected was uscal denwere fThe pr1999) C18 (Mtion atin the spectraUSA), t680 nmof the the Im

2.5. Co

A ssalar) versityanesthmassagwere mcontainin a prNaCl/L1 g/L, Mperformby injemountand quOnce tcontamwhich were tament, uwere asystemline veThrougunder under microsest posthe ma4 C.

2.6. St

The(SEM).observata samples were frozen with liquid nitrogen, sub-round and maintained at 4 C. Soon after, 20 mL ofater was added and rupture was performed by ultra-

a Misonix XL2000 Series sonicator, 10 pulses of 30 stervals until decomposition of the complex for all ofs. They were then incubated for 20 min at 30 C, beingd then ltered by gravity. Samples were then passedouble gauze lter and a No. 2 (125 mm) Wartman asy Fernndez et al. (2013). The nal extract was sub-easurement of polyphenols. Folin & Ciocalteu reagent

ollowing the protocol described by Lowry at 1951 Opti-was measured at 517 nm of the reaction. The samplesn and passed through HPLC to identify their prole.l described by Lohr and Wilhelm (Lohr and Wilhelm,modied, using the extracts for measurement with arey-Nagel, Duren, Germany) column, measuring reten-m. Then, the presence of diadinoxantina was identiedw fraction. The fresh samples were also observed in anfocal Olympus microscope (Olympus Fluoview 1000,mples were observed from an excitation of 280 nm toaining images of the stimulated structures. The imagesent channels and the DIC images were combined usingprogram.

ion and handling of semen samples

ion was made of adult male Atlantic salmon, (Salmong from sh farms associated with the Catholic Uni-emuco. Prior to the extraction of semen, sh wered with BZ-20 at 0.015%. Then, through an abdominal

stripping, the semen samples were extracted, whichained with oxygen, placed in hermetically sealed plasticand refrigerated. In the laboratory, semen was dilutedtion of 1:2 (semen: sperm diluent), composed of 18.8 g2, 3H2O 2 g/L KCl 72 g/L, NaH2PO4, H2O 4.1 g/L, NaHCO34, 7H2O 2.3 g/L and glucose 1 g/L. All procedures wereat 4 C. The samples were agitated and aerated daily

compressed oxygen. For observation, samples were a slide, adding 10 L of Atlantic salmon (S. salar) semen

mounted on the NIKON Labophot 2 optical microscope.mples were focused, 20 L of well water, river water,ed river water, and water with the Powermilt activatordiluted with distilled water (1), was applied. Protocol

to examine the time from the beginning of sperm move-all cells in the visual eld stopped moving, their kineticsed through a Computer Assisted Semen Analysis (CASA)he ImageJ program, curvilinear velocity (VCL), straighty (VSL) and straight line velocity (VAP) are described.is image analysis, the kinetic values were quantiedus conditions (Parodi et al., 2015). A video was made

zoom lens, as to generate a greater contrast with the in a lter phase, for the Nikon 10 lens and in the short-

time after the activation of sperm, in order to captureum amount of information. All solutions were kept at

cal analysis

lts are presented as the average standard error meanNOVA variance analysis was performed, comparing alls. A post-test was applied and the Bonferroni test was

104 P. Olivares et al. / Aquatic Toxicology 163 (2015) 102108

Fig. 1. Labora for madydimo water ymo. point of the sa

used for sepless than 0.0analysed w

3. Results

3.1. Mainte

A disadvlaboratory. be transporvivo modelFig. 1A, to with D. gemthese articthe two chano signicaorder to regcial rivers wshowed ex

Table 1Condition of adidymo artic

Parameters

Total volumUseful volumSlope Channel heigSubstratum Substratum pH T

O2

te. Uiver wry (o

showlabory of tory condition for didymo maintaining. A shows image of two articial river used , the number in the circles, show point of sample collection for observation of didmple collection.

aration of means with p < 0.05. Levels of probability (p)5 were considered statistically signicant. All data was

ith the Prism 4.0 statistical program.

geminacial rrecoveFig. 1Bin our viabilitnance model of D. geminata in the laboratory

antage of D. geminata, not have easy accessibility in theAlthough the D. geminata in conservation model canted, but there is no evidence of its cultured in the ex-. An articial river model was developed as shown inhave circulating river water and water contaminatedinata. River rocks with D. geminata were maintained inial rivers under conditions that were similar betweennnels, as summarized in Table 1 and Fig. 1B, which shownt physicochemical changes between the channels. Inister whether D. geminata are present or not, the arti-ere monitored over three observation points. Fig. 1A

ample of articial river used for maintaining the D.

rticial rivers. Show a table of the physical condition in control andial rivers system.

measured Control Didymo

e 28 (lt) 28 (lt)e 16 (lt) 16 (lt)

1.50% 1.50%ht 11.6 (cm) major, volume 766 (ml) 766 (ml)minor, volume 9.6 (ml) 9.6 (ml)

7.3 7.215 16150 143

nance in thdays of mogeminate. Tof 50% of DD. geminata

3.2. Effects

The micagent, althorivers throuliterature. Sies do not sulabile cells wet al., 2011)no increaserivers contarivers, mainof the micrconditions.cell death inprocess what that mompresence ofactivation tthe sampletozoa in waintaining dydimo in the lab upper panels control water lower panelB shows a time course of the number of didymo live in the different

pper panel, shows a control river and lower panel arti-ith D. geminata, the number indicate point of sample

bservation point) for D. geminata viability evaluation.s a plot graph of time course of viability of D. geminata

atory. At 0 days, when the experiment beginning 90% ofthe D. geminata are observed. After 50 days of mainte-

e laboratory 50% of the microalgae survived for 40 lastnitoring we observed a reduction of the live form of D.his suggests that it is possible to maintain viable values. geminata for months, which can be used as a source of

for laboratory studies.

of D. geminata on spermatozoa viability and function

roalgae, D. geminata, has not been identied as a toxicugh it has been suggested that it alters the ecology ofgh mechanisms that are unclear or not described in thetudies at the cellular level have not yet begun, and stud-ggest cytotoxicity. Fish spermatozoa in general are veryith high sensitivity to environmental changes (Cabrita

. The effect on their viability, as shown in Fig. 2A, indicate in the number of dead cells when exposed to water fromminated with D. geminata, nor to water from articialtained in the laboratory. Fig. 2B shows a quanticationophotographs and no signicant changes in any of the

These ndings suggest that didymo does not induce this model. S. salar spermatozoa undergo an activationen subjected to osmotic shock and present movementent which can last a few minutes. The effects of the

D. geminata on spermatozoa were explored, recordingime through the ImageJ-CASA program. The time thats spend moving was recorded. We observed sperma-ter with D. geminata to have 50% reduction in activity

P. Olivares et al. / Aquatic Toxicology 163 (2015) 102108 105

Fig. 2. Sperm absence (rivertion in the gu9 different exp

when compstudy, evalugeminata onFig. 3B showin control cmilt and wevidence thdays. Intereoratory werthey showeof conservasperm sampgeminata, anisms. Powosmotic shoseveral minto 1 with curve for Ponated riversthat the dilIC50 of 15 This evidenalters the acthe potentia

3.3. Observgeminata co

Using lowet al., 2015)VSL and VA

activation time, without affecting the viability of the samples. Eval-uations were made on whether the kinetics, another function ofspermatozoa, was affected by the presence of D. geminata. In Fig. 4A,a record of the traces of sperm movement is observed in the differ-

ivati the g sig

L andetic ty, a ta, as no eta cosed, idym

lyphle

wn mm ofoxancal e

phyed bgy, tle de

phar Polyols d

micc cont achextracells viability exposed to didymo. In A image of the sperm cells in the) or presence of didymo (river/didymo) In B a graph bar of quantica-re A from cell viability. The microphotographes are representative oferiment in different condition and the bar show means SEM.

ent actshowsshowinfor VSthe kinmobiligeminathere igeminadecreathat di

3.4. Pomolecu

Brothe fordiadinbiologion celldescribmacolomolecuual of 1948).protoced fororganicontenThese ared to uncontaminated river water. To complete thisations were made of the effects of the presence of D.

activation during the retention period of the samples.s a progression curve for the effect of time on viability

ells, which are activated with river water with Power-ater contaminated with D. geminata. The gure showsat the samples are viable after being conserved for 20stingly, when samples that were conserved in the lab-e activated with water contaminated with D. geminata,d a signicant decrease in activation times after 5 daystion. This suggests that after days of conservation of theles, they become more sensitive to the presence of D.

nd their viability can be affected by undened mecha-ermilt, a commercial solution (Figueroa et al., 2013) andck activates S. salar spermatozoa motility which lastsutes. In the samples, this was diluted from its stock 10deionized water. We constructed a V/V concentrationwermilt diluted with water from D. Geminata contami-, noting that there is a type of dose-response effect andution of Powermilt with water reduces this effect with

0.7 for D. geminata activation time as shown in Fig. 3C.ce suggests that water contaminated with D. geminatativation time of spermatozoa samples, even inhibitingting effect of commercial solutions such as Powermilt.

ation of spermatozoa kinetics in the presence of D.ntaminated waters.

-cost protocols implemented in the laboratory (Parodi kinetic changes were observed, particularly of the VCL,P values. The previous data indicated a change in the

activation tcentrationstime is 10 that D. gemrelease of peffect whenfunctions. Tgeminata afrivers.

4. Discussi

Evidencinata on thobserved owithout affmatozoa kicontent pretaminated wproblem inrivers in thleast three mjections areNo evidencdespite rapiis possible tit is not fea1988). A mriver substrof viable fotained for mon conditions using the CASA ImageJ program. Fig. 4Bobservation of the VCL in the various conditions, notnicant changes in samples. Fig. 4C and D show graphs

VAP where no signicant changes were observed inparameters either, but when assessing the progressivereduction of 50% was observed in the presence of D.s summarized in Fig. 4E. These experiments suggest thatffect on the kinetics of spermatozoa activation when D.ntaminated water was used. Although activation timet did not seem to affect motility. This evidence indicateso alters correct spermatozoa function.

enols, presence and possible D. geminata effector

icroalgae, including didymo, contain antioxidants in polyphenols (Lohr and Wilhelm 1999). A presence oftina is described in this type of microalgae, with diverseffects. Polyphenols have recognized benecial effectssiology, but toxic effects have also been reported. Asy Paracelsus, a classic physician and researcher in phar-he benecial or toxicological effects of a compound orpend on the concentration used, as quoted in this man-

macology and toxicology from Dr. Eichlotz (Eichholtz,phenols were extracted from D. geminata samples, usingescribed in (Fernndez et al., 2013), which were modi-roalgae. Fig. 5A displays a confocal image, which showstent of D. geminata in red and green. Fig. 5B showsieved in ppm from various sources of D. geminata.cts were used in experiments where we observed theime of spermatozoa when exposed to increasing con-

of the extract, nding that an IC50 dose of activation 0.7 ppm, as shown in Fig. 5C. Our ndings suggestinata can reduce the activation time because of the

olyphenols into rivers and that there is a doseresponse this type of extract is used in the S. Salar spermatozoahis suggests that there are mechanisms by which D.fects the correct operation of closed systems such as

on

e suggests an effect of water contaminated with D. gem-e motility time of S. salar spermatozoa. A decrease isf the activation time and percentage of motile cells,ecting the viability of the spermatozoa nor motile sper-netics. These effects could be mediated by polyphenolsent in D. geminata and can be released into rivers con-ith this microalga. D. geminata has become a pollution

the rivers of southern Chile. Its detection started ine Los Lagos Region, but since then, there have been at

ore regions reported that contain this plague and pro- that it will continue to spread if barriers are not raised.e exists that it can be maintained in a cultivated system,d growth (Sundareshwar et al., 2011). On the contrary, ito nd research indicating that under various conditions,sible to keep D. geminata in closed systems (Czarnecki,odel of articial rivers was developed (Fig. 1A) usingates, in the form of rocks with D. geminata. A numberrms of D. geminata of over 50% were able to be main-ore than two months, as shown in Fig. 1C. This allowed

106 P. Olivares et al. / Aquatic Toxicology 163 (2015) 102108

Fig. 3. The didymo alter function of sperm cells. A shows bar graph of activation time of sperm cells, exposed to river water, powermilt, distillated water or water contaminatedwith didymo. In B, graph time activation, in samples conserved for several days, exposed to river water, powermilt or water contaminated with didymo. In C shows a curvedoses respons waterand the dot sh

for conservweeks and fels, the effedescribed, rreports of s

Our not ata (F

Fig. 4. Kineticbar graph of Vgraph of VSL inThe bar show e, the time activation of sample exposed to powermilt plus increased proportion of ow means SEM from 12 independent experiments. *p < 0.05 (ANOVA).

ation of D. geminata contaminated water for severalacilitated experiments in the laboratory. In other mod-cts of D. geminata in the micro ora of rivers have been

2014).zoa is geminaeporting changes mainly in aquatic species, but with noimilar effects in more complex forms (Richardson et al.,

as the masvation time

of sperm cells exposed to didymo. A shows a traces record of the sperm cells, exposed CL in absences or presences of water contaminated with didymo, in C bar graph of VAP in

absences or presences of water contaminated with didymo, in E bar graph of motility of smeans SEM from 12 independent experiment. *p < 0.05 (ANOVA). contaminated with didymo, with IC50 of 16 07 didymo V/V. The bar

ndings indicate that the viability of S. salar spermato-ffected when exposed to water contaminated with D.ig. 2B). However, the reaction time is affected, as well

s of motile spermatozoa (Fig. 3A). A decrease in acti-

of 50% was shown (Fig. 3A), altering the functions of

to river water, powermilt or water contaminated with didymo. In B, absences or presences of water contaminated with didymo, in D barample in absences or presences of water contaminated with didymo.

P. Olivares et al. / Aquatic Toxicology 163 (2015) 102108 107

Fig. 5. Polyph mage of concentratio dymoin C doses res of pexperiment.

spermatozomatozoa accompound average of 4active mechit was abletimes. We owith D. gemtion of (1water, in incermilt on acD. geminatainto river wreduction oout affectinstudy with (Dietrich etwater alterevated spermreduces thequality of thination, suc

The orgaantioxidantnoxantina. benecial tmaintainedthere is a rgeminata, ato explore tent to the fenols present in didymo extract and the effect over sperm cells. A shows confocal in of polyphenols obtained from fresh sample of didymo, dry didymo from river, di

ponse curve of polyphenols effect over time of activation with IC50 of 10 0,7 ppma. The Powermilt compound has been used as a sper-tivator, as described by Figueroa et al. (2013). Thisproduced an excellent response in our samples with an

min of activation (Fig. 3C). In order to understand theanism of D. geminata, observations were made whether

to inhibit the effects of Powermilt on the activationbserved that upon dilution with water contaminatedinata, of the Powermilt stock (10) to a working solu-) with distilled water plus contaminated D. geminatareasing proportions (Fig. 3D) a decreased effect of Pow-tivation was observed, inhibiting it fully when 100% of

was used. This suggests that the release of moleculesater, with D. geminata would be responsible for thef the activation time of the S. salar spermatozoa with-g the kinetics of these spermatozoa (Fig. 4). Anothermetals in water, showed effects on kinetic parameters

al., 2010), but our results showed the contaminatedd the activation processes, except for the already acti-atozoa. This suggests a complex mechanism which

number of cells being able to be activated, but not theis activation, a different mechanism of classic contam-h as metal in the waters.nic content of D. geminata has proved to be rich in

polyphenols common to brown algae such as diadi-This type of molecule has cellular effects, ranging fromo toxic, depending on concentration. In the samples

or recovered from rivers, it was possible to show thatatio of about 200 ppm of polyphenols per 10 g of D.nd that it is possible to be extracted (Fig. 5B). In orderwhether it is possible to associate this organic con-unctional effects of D. geminata, the spermatozoa were

activated win Fig. 5C, sactivation oconclude thgeminata is discard oth

Recent sin vertebramunities (Gremoval (Laof the microthat didymospermatozosibly due tondings indand their efity conditioevidence shin our riveragent that a

Acknowled

Jorge Paresearch pr278-2472 Dtranslation,provided bValdevenitozoa and Powmicroscopeof fresh didymo exposed to 280688 nm of excitation. In B, bar graph with extend protocol of extraction and didymo dry in the laboratory,olyphenol. The bar and dot show means SEM from 9 independentith increasing concentrations of the extracts, as shownhowing a doseresponse effect when inhibiting timef the samples with an IC50 of 15 ppm of polyphenols. Weat the effect observed in waters contaminated with D.mediated by the presence of polyphenols, but we cannoter mechanisms of action for altering cellular function.tudies have suggested more toxic effects of D. geminatates (Richardson et al., 2014) and in benthic river com-illis and Chalifour, 2010) or of the treatments used forrned and Kilroy, 2014). This information on the effectsalgae is just recently being explored. The data suggests

can have a particular effect on activation time of S. salara. This is evidence of alterations in cell physiology, pos-

the presence of polyphenols, but more importantly, ouricate that the effects of this plague are more complexfect should be closely watched new study about fertil-n of the sh in contaminated river are required and theow a hypothesis for explain sh population reductions. D. geminata should be considered as a toxicologicalffects cell function.

gements

rodi receives contributions from MECESUP 0804, thisogram was supported by technical assistance from UCTidy 2013. We are indebted to Professor Ian Scott for his

revision and editing. Language editing services werey www.journalrevisions.com. We are indebted to Dr.

from BIOACUI-UCT for providing samples of spermato-ermilt. We thank Dr. Romero for access to the confocal

.

108 P. Olivares et al. / Aquatic Toxicology 163 (2015) 102108

References

Alavi, S.M., Rodina, M., Viveiros, A.T., Cosson, J., Gela, D., Boryshpolets, S., Linhart,O., 2009. Effects of osmolality on sperm morphology: motility and agellarwave parameters in Northern pike (Esox lucius L.). Theriogenology 72, 3243.

Bergey, E.A., Cooper, J.T., Phillips, B.C., 2010. Substrate characteristics affectcolonization by the bloom-forming diatom Didymosphenia geminata. Aquat.Ecol. 44, 3340.

Cabrita, E., Ma, S., Diogo, P., Martinez-Paramo, S., Sarasquete, C., Dinis, M.T., 2011.The inuence of certain aminoacids and vitamins on post-thaw sh spermmotility: viability and DNA fragmentation. Anim. Reprod. Sci. 125, 189195.

Clearwater, S.J., Jellyman, P.G., Biggs, B.J., Hickey, C.W., Blair, N., Clayton, J.S., 2010.Pulse-dose application of chelated copper to a river for Didymospheniageminata control: effects on macroinvertebrates and sh. Environ. Toxicol.Chem. 30, 181195.

Czarnecki, D.B., 1988. The Loras college non-diatom fresh-water algal culturecollection. Cryptogamie Algol. 9, 203209.

Dietrich, G.J., Dietrich, M., Kowalski, R.K., Dobosz, S., Karol, H., Demianowicz, W.,Glogowski, J., 2010. Exposure of rainbow trout milt to mercury and cadmiumalters sperm motility parameters and reproductive success. Aquat. Toxicol. 97,277284.

Eichholtz, F., 1948. Pharmacology and toxicology. Off. of Military Govt. forGermany, Field Information Agencies Technical, British, French, U.S., n. p.

Figueroa, E., Risopatron, J., Sanchez, R., Isachenko, E., Merino, O., Isachenko, V.,Valdebenito, I., 2013. Spermatozoa vitrication of sex-reversed rainbow trout(Oncorhynchus mykiss): effect of seminal plasma on physiological parameters.Aquaculture 372, 119126.

Gillis, C.A., Chalifour, M., 2010. Changes in the macrobenthic community structurefollowing the introduction of the invasive algae Didymosphenia geminata in theMatapedia River (Qubec, Canada). Hydrobiologia 647, 6370.

Hatef, A., Alavi, S.M.H., Golshan, M., Linhart, O., 2013. Toxicity of environmentalcontaminants to sh spermatozoa function in vitroa review. Aquat. Toxicol.140141, 134144.

Hu, Y.A., Lu, J.C., Shao, Y., Huang, Y.F., Lu, N.Q., 2013. Comparison of the semenanalysis results obtained from two branded computer-aided sperm analysissystems. Andrologia 45, 315318.

Jellyman, P.G., Clearwater, S.J., Clayton, J.S., Kilroy, C., Blair, N., Hickey, C.W., Biggs,B.J., 2010. Controlling the invasive diatom Didymosphenia geminata: anecotoxicity assessment of four potential biocides. Arch. Environ. Contam.Toxicol. 61, 115127.

Fernndez, I.J., Navarrete, P., Parodi, J., Romero, F., Salvatici, R.S., 2013. Chileancrude extract of Ruta graveolens generates vasodilatation in rat aorta atsubtoxic cellular concentrations. Adv. Biosci. Biotechnol. 04, 2936.

Kilroy, C., Larned, S.T., Biggs, B.J.F., 2009. The non-indigenous diatomDidymosphenia geminata alters benthic communities in New Zealand rivers.Freshwater Biol. 54, 19902002.

Kime, D.E., Ebrahimi, M., Nysten, K., Roelants, I., Rurangwa, E., Moore, H.D.M.,Ollevier, F., 1996. Use of computer assisted sperm analysis (CASA) formonitoring the effects of pollution on sperm quality of sh; application to theeffects of heavy metals. Aquat. Toxicol. 36, 223237.

Korkina, L.G., De Luca, C., Kostyuk, V.A., Pastore, S., 2009. Plant polyphenols andtumors from mechanisms to therapies, prevention, and protection againsttoxicity of anti-cancer treatments. Curr. Med. Chem. 16, 39433965.

Larned, S.T., Kilroy, C., 2014. Effects of Didymosphenia geminata removal on rivermacroinvertebrate communities. J. Freshwater Ecol. 29, 345362.

Lohr, M., Wilhelm, C., 1999. Algae displaying the diadinoxanthin cycle also possessthe violaxanthin cycle. Proc. Natl. Acad. Sci. U. S. A. 96, 87848789.

Parodi, J., 2014. Motility viability, and calcium in the sperm cells. Syst. Biol. Reprod.Med. 60, 6571.

Parodi, J., Ramrez-Reveco, A., Guerra, G., 2015. Example use of low-cost system forcapturing the kinetic parameters of sperm cells in Atlantic Salmon (Salmosalar). Adv. Biosci. Biotechnol. 06, 6372.

Reid, B.L., Hernandez, K.L., Frangopulos, M., Bauer, G., Lorca, M., Kilroy, C.,Spaulding, S., 2012. The invasion of the freshwater diatom Didymospheniageminata in Patagonia: prospects strategies, and implications for biosecurity ofinvasive microorganisms in continental waters. Conserv. Lett. 5, 432440.

Richardson, D., Oleksy, I., Hoellein, T., Arscott, D., Gibson, C., Root, S., 2014. Habitatcharacteristics, temporal variability, and macroinvertebrate communitiesassociated with a mat-forming nuisance diatom (Didymosphenia geminata) inCatskill mountain streams. N. Y. Aquat. Sci. 76, 553564.

Rivera, P., Basualto, S., Cruces, F., 2013. On the diatom Didymosphenia geminata(Lyngbye) M. Schmidt its morphology and distribution in Chile. Gayana Bot. 70,154158.

Root, S., OReilly, C.M., 2013. Didymo control: increasing the effectiveness ofdecontamination strategies and reducing spread. Fisheries 37, 440448.

Sundareshwar, P.V., Upadhayay, S., Abessa, M., Honomichl, S., Berdanier, B.,Spaulding, S.A., Sandvik, C., Trennepohl, A., 2011. Didymosphenia geminata:algal blooms in oligotrophic streams and rivers. Geophys. Res. Lett. 38, 15.

Takei, G.L., Mukai, C., Okuno, M., 2012. Transient Ca2+ mobilization caused byosmotic shock initiates salmonid sh sperm motility. J. Exp. Biol. 215, 630641.

Ubilla, A., Valdebenito, I.N., 1792. Use of antioxidants on rainbow troutOncorhynchus mykiss (Walbaum, sperm diluent: effects on motility andfertilizing capability. Lat. Am. J. Aquat. Res. 39, 338343.

Vladic, T.V., Jarvi, T., 2001. Sperm quality in the alternative reproductive tactics ofAtlantic salmon: the importance of the loaded rafe mechanism. Proc. Biol. Sci.268, 23752381.

Water contaminated with Didymosphenia geminata generates changes in Salmo salar spermatozoa activation times1 Introduction2 Methods2.1 Collection of D. geminata samples2.2 Protocol for maintenance of the D. geminata samples2.3 Mortality studies of D. geminata2.4 Extraction of polyphenols and confocal images2.5 Collection and handling of semen samples2.6 Statistical analysis

3 Results3.1 Maintenance model of D. geminata in the laboratory3.2 Effects of D. geminata on spermatozoa viability and function3.3 Observation of spermatozoa kinetics in the presence of D. geminata contaminated waters.3.4 Polyphenols, presence and possible D. geminata effector molecule

4 DiscussionAcknowledgementsReferences