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Butyl benzyl phthalate affects shoaling behavior and bottom-dwelling behavior in threespine stickleback

In this laboratory experiment, the effects on fish behavior caused by butyl benzyl phthalate (BBP) were of interest. We showed that shoaling behavior and bottom-dwelling behavior in threespine stickleback, Gasterosteus aculeatus, were altered as a
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   To whom correspondence should be addressed. Fax: # 47 7169 53 01. E-mail: asa.wibe @ Research Section A   89,  180 } 187 (2002)doi:10.1006/enrs.2002.4360 Butyl Benzyl Phthalate Affects Shoaling Behavior and Bottom-DwellingBehavior in Threespine Stickleback  A     s  sa Espmark Wibe, *  -   Anna Billing, *  Gunilla Rosenqvist, *  and Bj + rn Munro Jenssen * *Department of Zoology, Norwegian University of Science and Technology, 7491 Trondheim, Norway; and  -  Institute for Aquaculture Research, Akvaforsk AS, 6600 Sunndals ~ ra, Norway Received June 21, 2001 In this laboratory experiment, the effects on  A shbehavior caused by butyl benzyl phthalate (BBP)were of interest. We showed that shoaling behaviorandbottom-dwellingbehaviorinthreespinestickle-back,  Gasterosteus aculeatus  , were altered as aresult of exposure to 0.1 mg   / L BBP. Threespinesticklebacks, collected from a freshwater popula-tion in central Norway, were exposed to BBP for 26days. BBP was administered daily through thewater. We found that exposed  A sh aggregated moreinto one single shoal than control  A sh. Further, theexposed  A sh spent more time at the bottom of the test aquarium than the control  A sh. From theseresults we conclude that the behavior traitsaggregation and bottom-dwelling activity may besuitable and sensitive in detecting effects of BBP inthreespine stickleback.    2002 Elsevier Science (USA) Key Words: Gasterosteus aculeatus  ; shoaling be-havior; bottom-dwelling behavior; BBP; biomarker.INTRODUCTION Shoaling behavior and bottom-dwelling behaviorof  7 sh have to someextentbeen usedas indicatorsof pollution (Weis and Weis, 1974; Besch  et al ., 1977; Vogl  et al ., 1999; OConnor  et al ., 2000; Wibe  et al .,2001). Shoaling is de 7 ned as  7 sh staying in a groupfor social reasons, where structure of the group isless important. Schooling behavior additionally re-fers to the structure of the group, where 7 sh swiminapolarizedmanner(Pitcherand Parrish,1993).Onefunction of shoaling and schooling is to avoid attackfrom predators, since a predator will  7 nd it dif  7 cultto select one individual out of many (Pitcher andParrish, 1993).Tostayatthebottommaybearesultofmotionlessresting (e.g., OConnor  et al ., 2000) or it may be anindication of stress (Israeli-Weinstein and Kimmel,1998).Even though bottom-dwellinghas been inves-tigated in several studies (e.g. Vogl  et al ., 1999;Grillitsch  et al ., 1999; OConnor  et al ., 2000; Wibe  etal .,2001)thediscussionofwhetherthebehaviorisaconsequenceofstressorrestingmaybeinfrequent.Threespine sticklebacks,  Gasterosteus aculeatus ,often form shoalsthat in the breeding seasonconsistof females alone or a mixture of females and nonrep-roductive males (Whoriskey and FitzGerald, 1994).Throughouttherestoftheyearshoalsmayconsistof both males and females (Wootton, 1984). It has beenshown that shoals often consist of individuals of approximately the same size. This increases thehomogeneity of the shoals, which is considered anef  7 cient antipredator adaptation(Ranta  et al ., 1992;Peuhkuri  et al ., 1997). Shoaling may also increaseforaging success since each individual can spendmore time foraging instead of looking for predators,and the probability of   7 nding food will increasewhen more individuals are searching  (Pitcher andParrish, 1993; Peuhkuri  et al ., 1997).Butyl benzyl phthalate (BBP) is a phthalate esterthat is mainly used to increase the  8 exibility andworkability of plastic products such as toys, packag-ing materials, and vinyl  8 oors. Phthalates are pro-duced in large quantities. Additionally, since thecompounds are not chemically bound to the polymermatrix and thus may migrate from the plastic andinto the environment (e.g. Adams  et al ., 1995), thereis an increasing interest in the environmental fateand toxicology of phthalates. Signi 7 cant concentra-tions of BBP and other phthalates are found in foodsamples and in food packaging materials (Page andLacroix, 1995). BBP concentrations of 0.05 } 0.8  g/g have been found in food items (Page and Lacroix,1995), and in Norway, concentrations of 1.8   g/L 180 0013-9351/02  $ 35.00  2002 Elsevier Science (USA) All rights reserved.  have been detected in municipal waste waters (SFT,1998). As a comparison, the acute toxicity of BBPin  7 sh varies between 731 and 6470 ppb (Mayer  et al. , 1972; Adams  et al. , 1995).BBP may be metabolized to mono butyl phthalate(MBuP), mono benzyl phthalate (MBeP), hippuricacid, phthalic acid, benzoic acid, and an   -oxidizedmetabolite.Some of these metabolitesare suggestedto be toxic (Ema  et al ., 1995; Nativelle  et al ., 1999;Parkerton and Konkel, 2000). Observed effects of BBP include reproductive disorders in  7 sh and rats,suggestedas a result of possibleestrogen-mimicking properties of the phthalate (Jobling   et al ., 1995;Sharpe  et al ., 1995). Furthermore, altered feeding behavior in rats (Ema  et al. , 1991) and threespinestickleback (own unpublished data) and develop-mental and terratogenic effects in rats (Ema  et al. ,1995) are reported.Theaimof thisstudywastoexaminethein 8 uenceof BBP on shoaling and bottom-dwelling behaviorin threespine sticklebacks. In two different experi-mental setups, the differences between BBP-exposed  7 sh and control  7 sh in their ability to formshoals and to show preference for individuals of thesame size were investigated. Also the difference inbottom-dwelling behavior in control and BBP-exposed  7 sh was examined. METHODS  Fish Maintenance Threespine sticklebacks of two distinct sizeclasses, small (  N  " 211, average size " 4.54 $ 0.34 cm) and large (  N  " 160; average size " 5.63 $ 0.48), were captured with Plexiglas fry traps(Dolmen, 1982) in the small freshwater lakeKindsethtj + nna, S + r-Tr + ndelag county, central Nor-way (63.5 3 25   N, 10 3 45   E), in October. To minimizeexternal infections, the  7 sh were disinfected withNaCl (100 g NaCl in 10 L of water) for 15 min beforetransportation in plastic containers to Bratt + raResearch Centre, Norwegian University of Scienceand Technology, Trondheim, Norway.In the laboratory the  7 sh were divided into eightgroups: two control groups with small  7 sh(  N  " 53 #  N  " 53), two control groups with large 7 sh (  N  " 38 #  N  " 39), two BBP-exposed groupswith small  7 sh (  N  " 52 #  N  " 53), and two BBP-exposed groups with large  7 sh (  N  " 42 #  N  " 41).The respective groups were distributed betweeneight aquaria (85 L). The aquaria were equippedwith gravel, water plants,  Elodea sp ., and a station-ary water system with air supply. Daily, 25 %  of thewater in the aquaria was exchanged with de-chlorinated tap water contaminated with BBP andacetone (exposure groups) or acetone only (controlgroups).The 7 shwerefed daily ad lib with frozenmosquitolarvae. In the laboratory the temperature was keptat 5 } 7 3 C and the photoperiod was 9L:15Dh, whichare equivalent to the natural conditions for theactual season and location.  Exposure The exposure to BBP started 1 week after the  7 shhad arrived at the laboratory. A BBP stock solutionwas prepared using acetone as solvent (10 g BBP/Lacetone). A sample of the stock solution (0.85 mL)was daily added to the aquaria water. This gave anexposure concentration of approximately 0.1 mg/Las the total volume in each aquarium was 85 L. Theexposure continued for 26 consecutive days in Octo-ber and November. The control  7 sh were given thesame treatment as the exposed  7 sh, but only anequivalent amount of acetone without BBP was ad-ded to the daily exchanging water. Shoal Choice Experiment Ten minutes prior to each trial in the shoal choiceexperiment, test  7 sh were transferred from theirhome aquaria (85 L) to a test aquarium (38 L)(Fig. 1a). The test aquarium was equipped witha stationary water system and a thin layer of gravelto prevent stress among the  7 sh. The test aquariumwasdividedintothreecompartmentsseparatedwithtwo 7 xed glasswalls. The transferof the shoalsfromthe 85-L aquarium to the smaller 38-L test aquar-iumdidnotresultindetectablestresstoeithersmallor large  7 sh. After a few minutes in the test aquar-ium the  7 sh swam in the same manner as in thelarger home aquaria.In the shoal choice experiment, 30 trials with con-trolindividualsand32trialswith BBP-exposedindi-vidualswereconducted.Eachtrialconsistedofatestshoal of 15 large  7 sh in compartment ‘‘A,’’ a testshoal of 15 small  7 sh in compartment ‘‘B,’’ and onelarge focal  7 sh, de 7 ned as the choosing   7 sh in com-partment‘‘C’’ (Fig. 1a).It haspreviously been shownthat large  7 sh are more selective than small  7 shwithrespecttoassociationwith 7 shofcorresponding size (Ranta  et al ., 1992). Therefore, to be able todetect a change in choice between control and ex-posed 7 sh,thefocal 7 shwereofsizesimilartothatof the  7 sh in the large  7 sh group. The size of large andsmall shoal  7 sh in each trial did not overlap. Thelocation of the small and large test shoals were EFFECTS OF BUTYL BENZYL PHTHALATE ON BEHAVIOR OF STICKLEBACKS  181  10 cm 10 cm13 cm 13 cm25 cm25 cmA BDCC A  C B A B25 cm25 cm10 cm 51 cm (a)(b) 61 cm 61 cm FIG 1.  Experimental design. (a) Shoal choice experiment: A test shoal of 15 large and 15 small  7 sh were placed in compartments ‘‘A’’and‘‘B,’’ respectively.Afocal 7 shwasintroducedintocompartment‘‘C’’whereitwasallowedtochoosebetweencompartment‘‘A’’ (thefocal 7 sh stayed in C  ) and ‘‘B’’ (the focal  7 sh stayed in C  ). The solid lines represent permanent glass walls, while the dotted lines representimaginablechoicezones.A choice isde 7 nedas when the headofthe focal 7 sh crossesthe dottedline.Compartment‘‘D’’ istheneutral zonewhere no choice was recorded. (b) Size-assortative shoaling experiment: The aquarium was divided into two compartments, ‘‘A’’ and ‘‘B’’. A shoal containing 5 small and 5 large  7 sh was placed into compartment ‘‘A.’’ After the glass wall (solid line) was raised and the 7 sh wereallowed to swim in ‘‘A’’ and ‘‘B,’’ the shoaling behavior was recorded. randomizedbetweentheleftandtherightsideof thetest aquarium during the experiment. After the acclimation period of 10 min (Ranta andLindstro   K m, 1990) when the  7 sh seemed to behave intheir srcinal manner, the time that the focal  7 shspent in front of either shoal, i.e., large  7 sh (C  ) orsmall  7 sh (C  ), was recorded. Each trial was videorecorded for 10 min for further analyses. The videocamerawas placed1 m in frontof the testaquarium,and the operator left the room immediately afterstart of video recording. All  7 sh were allowed to seeeach other during the entire trial, including the ac-climationperiodof 10min beforethe video recording started. A choice was de 7 ned as when the head of the focal 7 sh crossed the choice zone (dotted lines in Fig. 1a).The neutral zone (‘‘D’’) was de 7 ned as no choice. Achoice furtherrequiredthatthefocal 7 shstayedinopen water and not at the bottom. Time spent at thebottom was also recorded. Each focal  7 sh was usedonly once, and one third of the 7 sh in each test shoalwere exchanged between each trial. To avoid reuse,the  7 sh were transferred to a separate aquariumafter each trial.182  WIBE ET AL.  Size-Assortative Shoaling Experiment The aim of the size-assortative shoaling experi-ment was to investigate whether there was a differ-ence between BBP-exposed  7 sh and control  7 sh intheirabilitytosegregateintosize-assortativeshoals.Ten minutes prior to each trial,  7 ve small and  7 velarge sticklebacks were transferred from their homeaquaria to compartment ‘‘A’’ in the test aquarium(38 L) (Fig. 1b). The test aquarium consisted of twocompartments (‘‘A’’ and ‘‘B’’) separated by a remov-able glass wall. The test aquarium was furtherequipped with a stationary water system and a thinlayer of gravel. In the size-assortative shoaling ex-periment, 30 trials with control individuals and 30trials with BBP-exposed individuals were conduc-ted. The size of the small and large  7 sh in each trialdid not overlap. After the acclimation period of 10 min (Ranta andLindstro   K m,1990)theglasswallwascarefullyraised,and the distribution of the  7 sh was recorded onceevery minute for 10 min, i.e., whether all 10  7 shaggregatedin one shoal or whether they were separ-ated into two or more shoals. Two shoals were de- 7 ned when two to eight individuals separated fromthe rest of the shoal. Individuals who were locatedless than one body length apart from each otherbelonged to the same shoal. Size-assortative shoalswere formed when the size-mixed shoal divided intoshoals consisting of individuals of the same size.With the exception of 60 %  of the  7 sh, each indi-vidual was only used once. The  7 sh used twice weregiven a restoration time of 3 days between the  7 rstand the second time. It was considered important toavoid having the retested  7 sh be put in a shoalwith  7 sh that they had already met. After each trialthe tested 7 sh were put in separate aquaria, to keepthetestedandthenottested 7 shapart.Thebehaviorwasvideo recorded for later analysesusing the samemethodasdescribedfortheshoalchoiceexperiment. After termination of the size-assortative shoaling experiment, the  7 sh were killed. A random sample(  N   " 40,  N   " 40) was sex-determined byautopsy. Chemical Analyses  After termination of the experiments most of the  7 sh were stored ( ! 20 3 C) before they weretransported frozen with air cargo to the NorwegianInstitute for Water Research in Oslo, Norway, foranalyses of   7 sh tissue BBP concentrations.To obtain suf  7 cient material for analyses several 7 sh were pooled (adding up to 10 g) and homogen-ized, before fat and fat-soluble pollutants were ex-tracted with dichlormethane in an ultrasonic bath.The extraction was cleaned on an ALOX column according to EPA method 606 (EPA, 1984).Di-allyl phthalate was added to the extraction asa recovery standard, while phenantrene was addedas an internal standard. The extraction was ana-lyzed using a gas chromatography (GC), HewlettPackard (HP) Model 5890 Series II, connected toa HP 5970 MSD instrument. The GC was equippedwith an on-column injector and a capillarycolumn type DB-5 (length 60 m, i.d. 0.25 mm,  7 lmthickness 0.25   m). BBP was identi 7 ed andquanti 7 ed according to retention time and masspeak signals. Statistics Forstatisticalanalysestwo-tailedtestswereused.Means are presented as medians and interquartileranges. Data were analyzed with the software SPSS(version 9.0), unless stated differently.Whether a  7 sh shoal segregated or not may beconsidered a binomial process. Thus, in order to testwhether exposed  7 sh differed from control  7 sh intheir probability of segregating from one to twoshoals with time (1 } 10 min), a multiple logisticregression analysis was applied by using theGENMOD procedure (using a logit link function,SASInstituteInc.,1996).Theprocedure 7 ttedagen-eralized linear model to the data by the use of max-imum likelihood techniques (e.g., McCullagh andNelder, 1989). Accordingly, as explanatory vari-ables, treatment (i.e., control or exposed) was in-cluded as a factor, whereas time (1 } 10 min) wasincluded as a continuous covariate. To account fora correlation in time from 1 min to the next, anothercovariate,  P t ! 1  was included in the model. This vari-able was calculated both for the control group andfor the exposed group, as  P t ! 1 "  A  /   N  , where  A  wasthe number of events (i.e., shoal segregation) inthe previous time step, and, correspondingly,  N   wasthe number of trials (  N  " 30) in the previous timestep. RESULTS  Autopsy of a randomly selected sample of smalland large  7 sh showed that there was no bias in sexratio between the two size groups that were used. Among the large 7 sh (  N  " 40) there were 19 femalesand 21 males. Among the small  7 sh (  N  " 40) thenumber of females and males were also 19 and 21,respectively. EFFECTS OF BUTYL BENZYL PHTHALATE ON BEHAVIOR OF STICKLEBACKS  183  TABLE 1  Average (Median with Interquartile Ranges) Amount of Time That the Control and Exposed Focal Fish Spent inFront of Shoals Containing Small or Large Fish, in theNeutral Zone, or at the Bottom Control   Exposed   StatisticsTime (min) infront of large 7 sh (C  )2.29  (0.46 } 7.61)3.27  (0.00 } 5.40) U  " 414, df  " 61,  P " 0.35Time (min) infront of small 7 sh (C  )4.42  (1.19 } 7.45)1.74  (0.00 } 4.96) U  " 353, df  " 61,  P " 0.07Time (min) inneutral zone(D)1.84  (0.69 } 4.07)0.48  (0.29 } 0.99) U  " 279, df  " 61,  P " 0.005Time (min) atthe bottom0  (0.00 } 0.00) 1.03  (0.00 } 7.04) U  " 243, df  " 61,  P ( 0.001  Note . Intergroup comparisons are tested with Mann } Whitney U   test; intragroup multiple comparisons are tested with Krus-kal } Wallis (Dunn).  Comparable medians with different superscript letters aresigni 7 cantly different (  P  0.05). TABLE 2 Differences between Controls and BBP-Exposed Fishin Average Observed Time in Front of Either Shoal withSmall or Shoal with Large Fish Proportion of observed time inC  , C  , or D Control Exposed StatisticsC  # C   /C  # C  # D0.82(0.59 } 0.93)0.92(0.71 } 0.96) U  " 392,df  " 61,  P " 0.21Expected proportion of time (0.73)  (C  # C   /C  # C  # D)Control:One-sample  t  test:  t " 0.32,  df  " 29,  P " 0.8Exposed:One-sample  t  test:  t " 0.53,  df  " 31,  P " 0.6  Note . There were no difference in time spent in C   (time spentin front of large  7 sh) or C   (time spent in front of small  7 sh) andtime spent at random locations for either control focal  7 shor exposed focal  7 sh. Expected proportion of time(0.73) " (C  # C   /C  # C  # D) (D " neutral zone). Results aretested with Mann } Whitney  U   test (mean expressed as medianwithinterquartileranges) and one-sample t  test. C  , time in frontof large  7 sh; C  , time in front of small 7 sh; D, time in the neutralzone. a Expected proportion of time (0.73) " (C  # C   /C  # C  # D)cm (Fig. 1a) Shoal Choice Experiment Exposedfocal 7 sh spent more of the observed timeat the bottom of the test aquarium than the controlfocal  7 sh (Mann } Whitney  U   test:  U  " 243,  N  " 62,  P ( 0.001). Exposed focal  7 sh also spent less of theobserved time in the neutral zone (D) than controlfocal  7 sh (Mann } Whitney  U   test:  U  " 279,  N  " 62,  P " 0.005) (Table 1).There were no differences between control andexposed focal  7 sh in the time spent in front of eitherthe shoal of large  7 sh (C  ) or the shoal of small  7 sh(C  ) (Fig. 1a) (time spent in C  # C   /C  # C  # D)(Mann } Whitney  U   test:  U  " 392,  N  " 62,  P " 0.21)(Table 2).There were no difference in time spent in C   orC   (Table 1) and time spent at random locations foreither control focal  7 sh or exposed focal 7 sh (propor-tion of time expected by chance " C  # C   /C  # C  # D " 0.73 (Fig. 1a)) (exposed [one sample t  test:  t " 0.317,  N  " 30,  P " 0.8], control [onesample  t  test:  t " 0.53,  N  " 30,  P " 0.6]) (Table 2).The intragroup multiple comparison showed thatexposed focal  7 sh spent less of the observed time inthe neutral zone than in C  , C  , or at the bottom(Kruskal } Wallis (Dunn):   2 " 18.5,  N  " 87,  P " 0.0003). The control focal  7 sh spent less time inthe neutral zone than in front of small  7 sh(  P " 0.04). There was no difference in their timespent in the neutral zone compared with their timespent in front of large  7 sh (  P " 1.00). Furthermore,the control 7 sh spentless of the observedtime at thebottomcomparedtoany otherplace(Kruskal } Wallis(Dunn):   2 " 32.91,  N  " 92,  P ( 0.01) (Table 1). Size-Assortative Shoaling Experiment Exposed  7 sh stayed in one shoal for a longerperiod of time than the control  7 sh (Fig. 2) (Mann } Whitney  U   test:  U  " 206,  N  " 60,  P ( 0.001). Ex-posed  7 sh also differed from control  7 sh in theirprobabilityof segregating from one mixed shoal intotwo shoals with time (1 } 10 min) (Fig. 3) (GENMODlogistic regression, treatment:  N  " 20,   2 " 40.38,  P ( 0.001; time:  N  " 20,   2 " 6.72,  P " 0.01). Chemical Analyses The analyses of BBP in  7 sh homogenate showedthat the concentrations in all exposed groups wereunder the detection limit (100 ng/g w.w.) for theinstrument. DISCUSSION This study showed that shoaling behavior andbottom-dwelling behavior in threespine stickleback184  WIBE ET AL.
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