influence of a xenobiotic mixture (pcb and tbt) compared to single substances on swimming behavior...

Acta hydrochim. hydrobiol. 33 (2005) 4, 287−300 287

Katja Schmidta, Influence of a Xenobiotic Mixture (PCB and TBT)Christian E.W. Steinberga,Georg B. O. Staaksa, Compared to Single Substances on SwimmingStephan Pflugmachera

Behavior or Reproduction of Daphnia magnaa Leibniz-Institute of Freshwater

Ecology and Inland Fisheries, Aroclor 1254, a technical PCB mixture (polychlorinated biphenyls) and TBT (tributyltin-Müggelseedamm 301, chloride) are environmental pollutants that cause a broad spectrum of acute toxic and12587 Berlin, Germany

chronic effects in aquatic animals. In this paper, the sensitivity of Daphnia magna tochronic exposure to mixed xenobiotics was evaluated under laboratory conditions. Theresults show that xenobiotic mixtures (50% each of the single compounds) were moretoxic than individual xenobiotics alone.By measuring behavioral parameters of animals, it becomes evident that exposure tosingle xenobiotics significantly affects daphnids: exposure led finally to a rapid decreasein mean swimming activity and also caused changes in preferred swimming depth, withdaphnids preferring the upper layers of aquaria. The mixture altered the swimming be-havior even more strongly compared to the group stressed by single chemicals. Finally,all daphnids sank to the bottom of the aquaria, still alive, but inactive at the end of theexposure period. In addition, we investigated the reproductive capacity (number ofnewborn per female and day). PCB did not affect the number of newborn significantly,TBT-stress led to an evidently decreased number of young daphnids and the xenobioticmixture decreased reproduction even more.In conclusion, we found significant effects of the single compounds as well as approxi-mately additive (swimming behavior) and synergistic (reproduction) effects of the chemicalmixture on daphnids indicating the possibility of dramatic ecological consequences of theoccurrence of mixed xenobiotic substances in the aquatic environment.

Einfluss einer xenobiotischen Mischung (PCB und TBT) auf Schwimmverhaltenoder Reproduktion von Daphnia magna

Aroclor 1254, eine technische PCB-Mischung (polychlorierte Biphenyle), und TBT (Tributyl-zinn) sind Umweltbelastungen, die ein breites Spektrum akut toxischer und chronischer Wir-kungen in aquatischen Tieren verursachen. Untersucht wird die Sensitivität von Daphniamagna bei chronischer Exposition gegenüber gemischten Xenobiotika unter Laborbedingun-gen. Die Ergebnisse belegen, dass xenobiotische Mischungen (50% jeder Substanz) stärkertoxisch wirken als jede Substanz für sich allein. Durch Messung von Verhaltens-Parameternder Tiere wird deutlich, dass die Exposition gegen einzelne Xenobiotika Daphnien signifikantbeeinflusst: Die Wirkung führt letztlich zu einer rapiden Abnahme der Schwimmaktivität undverursacht bei den Daphnien Veränderungen in der Aufenthaltstiefe gegenüber den sonstbevorzugten oberen Schichten eines Aquariums. Die Stoffmischung verändert dasSchwimmverhalten stärker als die Einzelstoffe: Zu Versuchsende sinken alle Daphnien le-bend, aber inaktiv zu Boden. Die zusätzlich untersuchte Reproduktionskapazität (Anzahl derNeonaten je Weibchen) zeigt: PCB beeinflusst die Zahl der Neonaten nicht signifikant, TBT-Stress führt zu einer deutlich verringerten Zahl junger Daphnien und die Mischung beiderStoffe senkt die Reproduktion noch stärker. Zusammenfassend wurden signifikante Wirkun-gen der Einzelstoffe beobachtet sowie additive (Schwimmverhalten) als auch synergistische(Reproduktion) Wirkungen der Stoffmischung. Dies indiziert die Möglichkeit dramatischerökologischer Konsequenzen aus dem Vorkommen gemischter xenobiotischer Stoffe in deraquatischen Umwelt.

Keywords: Polychlorinated Biphenyls, Tributyltin

Schlagwörter: Polychlorierte Biphenyle, Tributylzinn

Correspondence: K. Schmidt, E-mail: [email protected]

DOI 10.1002/aheh.200400579 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

288 K. Schmidt et al. Acta hydrochim. hydrobiol. 33 (2005) 4, 287−300

1 Introduction

Many aquatic ecosystems are faced with spatially or tem-porally alarming high levels and complex mixtures of xeno-biotics as a result of the transport from industrial areas intothe environment and their chemical persistence [1, 2]. Thecompounds studied in the present experiments were tributyl-tinchloride (TBT) and the PCB mixture Aroclor 1254, whichwere selected as typical man-made chemicals released intothe environment on purpose or by accident. TBT is widelyused in antifouling paints and therefore significant concen-trations of TBT can be measured in (natural) waterways.This has resulted in TBT concentrations up to 5.76 µg L�1

in Canadian freshwater, 1.5 µg L�1 in marine waters inFrance, and 7.2 µg L�1 in harbors in the Netherlands [3].TBT levels as high as 61.8 µg L�1 have been measured inGerman industrial effluent nearby Bremen [4]. Mixtures ofPCB isomers with different ingredients were used in mech-anical engineering and the chemical and electrotechnical in-dustries. Restrictions on the use of PCB have resulted inconsiderable reductions in surface water concentrations, butPCB are known to be highly persistent and therefore signifi-cant quantities are still detectable in most parts of the en-vironment , e.g. 9.6...567 µg g�1 (dw) in freshwater sedi-ments [5, 6]. PCB has been detected in the USA: in LakeMichigan (1980; 3.2 ng L�1) and in San-Francisco-Bay(1993�1995; 1.6 µg L�1) [7, 8].

Both TBT and PCB are toxic and a variety of effects on or-ganisms have been reported [3, 5]. Bioassays conductedusing survival and reproduction as measures of the relativetoxicity of different PCB-mixtures (Aroclor 1221, 1232, 1242,1248, 1254, 1260, 1262, 1268) showed Aroclor 1254 to bethe most toxic to Daphnia magna under continuous-flow con-ditions with a 3-wk LC50 of 1.3 µg L�1 [9]. PCB congeners(IUPAC numbers 52, 77, 101, 118, 138, 153, 180) had littleor no detectable sublethal effects on reproduction andgrowth: the number of neonates produced was either un-affected or enhanced [10]. TBT reduces reproductive per-formance, neonate survival, and juvenile growth rate in crus-taceans [11]. TBT proved to be acutely toxic to Daphniamagna with an LC50 value of 0.9 µg L�1 [12]. Production ofmetabolites of testosterone was elevated following exposureto 1.25 µg L�1 TBT [13].

Most of the recent research on the influence of xenobioticson daphnids has focused on the effects of single chemicalapplication and there are only a few in vivo studies on influ-ences of xenobiotic mixtures on animals [14, 15]. Presently,there are various models predicting the interactive effects ofchemicals; e.g. they can be antagonistic, additive or syner-gistic. The interactive effect of cyanide and trimethyltin onArtemia mortality was found to be antagonistic, in contrastto the synergistic effect of trimethyltin and beryllium [12].With the two models ‘concentration-addition’ and ‘independ-

2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

ent action’, a pragmatic way to analyze combined effects ispossible using experimental information on single substancetoxicity [16�18]. Most of the data with binare mixtures basedon the EC50 values of single substances, e.g. the interactionbetween several pesticides including TBT and Methyl-tert-butyl ether [19]. In natural environments, species are ex-posed to a large number of chemicals in lower concen-trations and, therefore, the present study investigates theeffects of a mixture using only 50% of each xenobiotic com-ponent.

The quantitative behavior analysis and the reproductiveability of aquatic animals proved to be appropriate bioindica-tors for stress situations. Different substances in a wide vari-ety of organisms can, for instance, change the locomotiveactivity of aquatic animals [20�22]. The swimming motion ofindividual Daphnia magna was continuously monitored andexposure to pesticides caused a significant increase in thefractional dimension [23]. Other results showed that cad-mium decreased the velocity of Daphnia magna [24]. In theabsence of pollutants, a clone (Daphnia magna) showedpositively phototactic behavior, and the behavior becamesignificantly less positive with increasing concentrations of achemical substance [25]. In our opinion, the behavior of ani-mals ensures ecological reactivity and adaptation to their(e.g. particularly toxic) environment. For permanently swim-ming zooplankters such as daphnids, swimming activity hasan especially great physiological and ecological relevance.

In general, the effects of xenobiotic stress on daphnid lifehistory include reduced or delayed reproduction and, conse-quently, the toxicity may affect population growth rates.Daphnids exhibit parthenogenesis, being capable of bothasexual and sexual reproduction; female offspring are pro-duced under optimal conditions such as normal temperatureand adequate food. Little is known about the biochemicalmechanisms involved in changing reproductive strategies inDaphnia magna [26]. Although estrogens and testosteronehave been isolated in Daphnia magna, their physiologicalroles during sex determination, including endocrine disrup-tion by environmental pollutants, have yet to be elucidated[27]. For example, in Daphnia magna, 4-nonylphenol hasbeen shown to produce metabolic androgenization [28].Therefore, we investigated the effects of PCB and TBTexposure and the possibility of synergistic or antagonisticeffects of mixed xenobiotics on the number of newborndaphnids.

The aim of our experiments was to observe chronic effectson daphnids caused by TBT and PCB (Aroclor 1254) or bymixture of both. Furthermore, the present study points toexposure induced shifts in energy allocation in the daphnidsthat change the available energy for locomotion and repro-duction in the presence of xenobiotic substances. The re-

Acta hydrochim. hydrobiol. 33 (2005) 4, 287−300 Xenobiotic Mixture and Single Compounds Influenced Daphnids 289

sults illustrate the importance of further investigations ofother direct and indirect effects, e.g. reduced consumptionprocesses, and increased oxygen consumption, which areclosely connected with trade-offs in chemically exposeddaphnids.

2 Materials and methods

2.1 Zooplankton

The single clone of Daphnia magna originated from a naturalpond (fish farm Kreba, Germany). Only 7 day old females(generally with developing embryos) were used for theexperimental series. The animals were kept in a syntheticmedium as described by Zehnder and Gorham [29] at(20 ± 3) °C under a 12 h : 12 h photoperiod using halogenlamps (60 Watt). The medium was renewed weekly and ani-mals were fed with living green algal cells (approximately50% Chlorella minutissima and 50% Scenedesmus acutus)once a day.

Food cells were maintained as a semi continuous cultureand were also grown in a synthetic medium according toZehnder and Gorham [29]. All culture suspensions were keptat a temperature of (20 ± 3) °C and under a light regime of12 h :12 h. Algal cells were collected during the log-growthphase; the cell density was counted using the photometricmethod of Kohl and Nicklisch [30] to make up food concen-trations of 100 mm3 biovolume per liter.

2.2 Chemicals

We used two xenobiotic substances: a technical PCB mix-ture (Aroclor 1254, Boeringer, Germany) and tributyltinchlo-ride (TBT, Sigma-Aldrich, Germany). The purity of the chemi-cals used was 96% for TBT and 98% for PCB. At the begin-ning of each experiment, dilutions of the chemicals were pre-pared using methanol (99.9% purity) as a solvent and storedat 4 °C. The final maximum MeOH exposure concentrationwas 5 µL L�1, which did not cause any significant effects onthe daphnids. Portions of the stock solutions were dilutedwith distilled water daily to obtain the final concentrationsused in tests:

� Single chemicals: PCB (15.0 µg L�1) and TBT(6.6 µg L�1)

� Xenobiotic mixture: 50% PCB (7.5 µg L�1) and 50% TBT(3.3 µg L�1).

Finally, we used 100% of the single chemicals in our basictests and correspondingly only 50% of each xenobiotic com-ponent for the subsequent mixture tests.


To measure the actual aqueous concentrations of PCB orTBT, the medium was collected from each experiment duringthe exposure period; the chemical analysis of these watersamples was carried out by means of GC/MS (organotin;DIN 38407-13) and GC/ECD (PCB; DIN 38407-3).

2.3 Experimental design

Automated biomonitoring is defined as systems that detecttoxic conditions on a continuous basis in whole organisms,in this case, with behavioral endpoints [31].

The swimming behavior of individual daphnids wasmeasured using the automated video imaging systemBehavioQuant (Metacom, Germany), which has been usedin various previous studies [21, 22, 32, 33].

Test aquaria were observed by video cameras and the posi-tions of each single animal were recorded. The chamberswere filmed at a speed of 0.04 s per frames and eachaquarium was studied for 2 min periods at 20 min intervalsover 23 hours from 10.00 a.m. to 9.00 a.m. The images weredigitized and the results of two minute periods were aver-aged into one read-out. The swimming parameters motility(swimming speed in video-pix s�1) and swimming depth be-low surface (swimming depth in pix) were analyzed.

The swimming space of each test chamber measured80 mm � 60 mm � 10 mm (length � depth � width). Thesedimensions were transferred automatically into the Behavio-Quant-units 0...250 pix (length) and 0...180 pix (depth).Tests chambers were designed as a through-flow system,with a solution renewal rate of 500 mL per day being takenfrom storage tanks with daphnid medium according toZehnder and Gorham [29]. The chemicals (PCB or TBT orboth) and food particles (50% Chlorella minutissima and50% Scenedesmus acutus at a concentration of approxi-mately 10 mm3 L�1) were added to the continually aeratedstorage tanks and the medium in the storage tanks was re-newed daily. Six animals were picked from the stock culturesand transferred to each test aquarium. The animals wereallowed to acclimate to the experimental conditions for ap-proximately one hour and were then observed withoutchemical stress (7 d non-exposure period) before being ex-posed to xenobiotic substances for 21 d. Only two of ourbehavioral tests (mixture and PCB exposure) were termin-ated at the end of the 13th exposure day, because daphnidsshowed pathological symptoms (particularly the absence orirregular frequency of antennal movement) and clear be-havioral changes due to the xenobiotics, still living. A tem-perature of (20 ± 2) °C and constant light (cold light source)were maintained throughout the experiment. Animals wereexamined daily and test chambers were cleaned mechan-ically and waste was removed. Once daily, the newborn


daphnids were removed, because only mature animals wereincluded in the calculations of swimming activity.

As a chronic toxicity parameter, we investigated repro-duction of the daphnids, which was carried out in 1 L aqua-ria. For each aquarium, 10 daphnids were collected from thestock culture; food and chemicals were added to the daph-nid’s medium as described above. During the whole exposureperiod of 21 days, a temperature of (20 ± 3) °C and a light-dark cycle of 12 h : 12 h were maintained. The animals wereexamined, each aquarium was cleaned mechanically, andwaste was removed daily. Additionally, newborn daphnidswere counted and removed daily and 50% of the medium(including food cells and xenobiotics) was exchanged.

Usually, two groups served as controls and the various expo-sure concentrations were examined in at least duplicates.The solvent used for the stock solutions did not affect repro-duction significantly.

2.4 Statistics

Behavioral data were expressed as means ± standard devi-ation over the 6 individuals for each measuring period of twominutes and these data were calculated as mean ± standarddeviation for 1 or 23 h, or for approximately 1 week. Thereproduction data were expressed as means ± standard de-viation over 3 groups (each 10 females) for each exposuregroup per day or over the whole exposure period. The sta-tistical differences in both behavioral variables and repro-duction results were assessed using the Mann and WhitneyU-test and the Wilcoxon Rang-test [34]. All statistical analy-ses were calculated with n indicating number of animals andcarried out at a 95% level of significance (p = 0.05).

Fig. 1: Mean motility of D. magna(n = 6) exposed to PCB (15 µgL�1), TBT (6.6 µg L�1), or a mix-ture of both (50% of each xeno-biotic chemical) compared to con-trol. A...G: non-exposure days;1...21: exposure days.

Mittlere Bewegungsaktivität vonD. magna (n = 6), exponiertgegen PCB (15 µg L�1), TBT(6.6 µg L�1) oder eine Mischungbeider (50% jeder Substanz) imVergleich zur Kontrolle. A...G:Tage vor der Exposition, 1...21Tage der Exposition.


3 Results and discussion

3.1 Swimming behavior

No mortality occurred in the experiments to assess theswimming behavior of D. magna under non-exposure con-ditions. Therefore, the results under non-exposed conditionswere taken as a standard swimming behavior. In all parallelexperiments, swimming activity was found to increaseslightly over 28 days, due to the development and bodygrowth of the daphnids. Methanol exposure did not causesignificant effects on the daphnids.

Figure 1 shows the mean swimming speed (motility) per dayof four different groups: control group without chemicals andthree exposure groups stressed by different xenobiotic expo-sures (day 1...21; PCB and TBT alone and the mixture expo-sure). The mean swimming speed per week of non-exposeddaphnids was found to be (14.6 ± 0.9) pix s�1 (day A...G),(16.4 ± 0.8) pix s�1 (day 1...7), (17.9 ± 1.7) pix s�1 (day8...13), and (20.6 ± 0.9) pix s�1 (day 14...21).

During the first exposure days (days 1...9), we did not findsignificant relationships between the xenobiotic exposureand swimming speed in any exposure groups. A substancerelated impact on daphnid activity was first observed on the10th exposure day: animals exposed to the xenobiotic mix-ture of 50% TBT and 50% of PCB showed a rapid decreasein mean motility. The PCB exposed daphnids began to de-crease their mean swimming speed on the 11th exposureday whereas, in contrast, TBT exposure first caused a de-crease in mean motility on the 19th exposure day.

We not only found differences in the time-dependent re-sponse of the exposed daphnids, but also in the amplitudeof changes of swimming which were significantly different


between the different exposures. The mixture caused a rapidreduction in swimming speed: daphnids showed a mean mo-tility without chemicals (days A...G) of (15.4 ± 0.6) pix s�1

decreasing to (17.7 ± 0.9) pix s�1 (days 1...7) and then to(13.7 ± 3.7) pix s�1 on days 8...13). Finally, daphnids ex-posed to the mixture showed a maximal reduction of 23.7%compared to the control. Exposure to PCB also led to a rap-idly decreased motility from (15.2 ± 0.7) pix s�1 (days A...G),to (15.3 ± 0.6) pix s�1 (days 1...7) falling to (11.8 ± 5.3)pix s�1 (days 8...13). The strongest reduction in swimmingspeed was noted during the last exposure week, with a 34%reduction compared to the control group. In contrast to PCBand mixture exposure, TBT exposure caused a slight in-creased in motility over the whole exposure period, as com-pared to the control group, but a sudden significant decreasein swimming speed was found from day 19 onward. The mo-tility of TBT-exposed daphnids was (14.5 ± 0.8) pix s�1 (daysA...G), (17.1 ± 0.7) pix s�1 (days 1...7), (19.3 ± 1.6) pix s�1

(days 8...13), and (19.7 ± 2.9) pix s�1 (days 14...21). Themaximum reduction of the swimming speed was 4.6% dur-ing the last week, as compared to the control.

The reaction of daphnids exposed to the mixture (23.7% de-creased motility) appears to be an additive effect, becausethe sum of the clearly decreased (PCB: 34%) and slightlyincreased (TBT: 7.7%) motility of the single exposure groupsresults in an effect in the same range (26.3% decreasedmotility during second exposure week) as for the mixturegroup.

No mortality occurred in any experiments investigating thepreferred swimming depth of daphnids exposed to single ormixed xenobiotics. Also, solvent exposure did not cause sig-nificant effects on the daphnids. Non-exposed animals pre-ferred deep layers near the bottom of the test chamber andthe mean daily swimming depth was noted to be non-syn-

Fig. 2: Mean swimming depth ofD. magna (n = 6) exposed to PCB(15 µg L�1), TBT (6.6 µg L�1), ora mixture of both (50% of eachxenobiotic chemical) comparedto control. A...G: non-exposuredays; 1...21: exposure days.

Mittlere Schwimm-Tiefe von D.magna (n = 6) exponiert ge-gen PCB (15 µg L�1), TBT(6.6 µg L�1) oder eine Mischungbeider (50% jeder Substanz) imVergleich zur Kontrolle. A...G:Tage vor der Exposition, 1...21Tage der Exposition.


chronized, ranging between minimum of (145.2 ± 4.3) pix(days 14...21) and a maximum of (156.9 ± 3.9) pix (days1...7). A typical reaction of daphnids under chemical stresswas a rapid decrease in the preferred swimming depth (toupper layers in the aquaria, near the surface) followed by anincreased sinking to the bottom of the aquarium. The signifi-cantly increased swimming depth to the bottom was corre-lated with an increasing number of daphnids with verysparse antennal movements.

The results of a single substance or mixture exposure testare given in Figure 2, which displays daphnids’ mean swim-ming depth in the experiment during the non-exposure per-iod (days A...G) and exposure times (days 1...21). The ear-liest reaction of daphnids was caused by the mixture expo-sure: daphnids preferred a swimming depth nearer to thesurface during the 8th...11th exposure days. PCB-exposeddaphnids preferred the upper layers during days 9...11, andthe TBT-exposed group showed a decreased swimmingdepth during days 10...13. The following rapid sinking todeeper layers of the aquaria among the single and mixtureexposed groups also proceeded in a time dependent manner:daphnids exposed to the mixture changed their behavior first(days 11...13), followed by PCB-exposed group (days12...13), with the TBT-exposed daphnids sinking last (day20...21), still living.

Changes in the swimming depth were not only time-depend-ent. Quantitative measurements also showed significant ef-fects of the xenobiotics on daphnids. The most decreasedswimming depth was found in daphnids exposed to the mix-ture, followed by PCB and TBT exposures. In no groups diddaphnids change their swimming depth during the first expo-sure week (days 1...7), but they clearly preferred the upperlayers of the aquarium during the second (mixture group:(120.2 ± 43.4) pix; PCB group: (135.4 ± 30.5) pix) and third


exposure weeks (TBT group: (124.1 ± 17.6) pix). The maxi-mum change in swimming depth was higher (20.6% nearerto the surface) in the mixture group than in the single sub-stance groups (TBT: 15.5% and PCB 10.6%) compared tothe controls. By comparison with the sum of the single expo-sure results (26.1%) we found that the effect of the mixtureon the preferred swimming depth was approximately addi-tive, since the values lie in the same range.

It should be pointed out that differences between individualsin the first reaction of daphnids led to high variability in theactivity or swimming depth and therefore, the standard devi-ation of mean motility and swimming depth were noted to bevery high, when daphnids showed the first significantchanges in their behavioral pattern (approximately 2ndexposure week). The xenobiotic mixture always caused thefirst changes in swimming behavior (8th and 10th days), fol-lowed by the PCB-exposed daphnids (9th and 11th days)and, in contrast, TBT led to effects on swimming pattern onthe 10th and 19th days. Generally it seems that PCB was amore toxic substance than TBT, but the mixture exposurewas found to cause the most adverse effects on daphnids.

The affected swimming behavior of daphnids was registeredfirst at the 9th exposure day with gradually increasingchanges in activity and swimming depth. Therefore, it seemsthat the toxic influence of chemicals cause a slow break-down of several biological functions instead of a specific dis-turbance of only one organ. Also, the effects of TBT on re-production and survival were found to increase with time, asshown in our study and by other authors [36, 37]. Similarresults have been described by other researchers, who alsofound a time-dependent increase in the effects of TBT onswimming velocity of cadmium-stressed daphnids [24].These findings could lead to the assumption, that toxic PCBand TBT are slowly taken into several cell-types resulting inan unspecific disruption of physiology. The causes of theselosses of biological function could be the inhibition or acti-vation of several enzymes, as described by various authors[13, 38�41] for PCB and TBT. Thus, it is very difficult todetermine a single cause of daphnid poisoning caused bythe chemicals used in our experiments. Additionally, in-hibited movement could lead to a destruction of populationbehavior, such as diel vertical migration, possibly leading toincreased predatory pressure, or uncomfortable environ-mental conditions (e.g. food sources). Therefore, even a lowconcentration of chemical could lead to several sublethal(decreasing population growth) or even toxic (mortality) ef-fects.

All disturbances of the physiological steady state of animalslead to adaptive responses and have more or less pathologi-cal effects, which are doubtlessly energy demanding. Also,behavioral changes in daphnids such as those described


could be caused by a disturbed physiology. The results pre-sented here indicate that chemical exposure caused cleardecrease in swimming speed. We hypothesized that the in-hibited swimming activity (muscular movement) may becaused either by a lowered main energy budget or by anelevated energy demand of other physiological processes(activity of enzymes involved in biotransformation pro-cesses). Hence, the preferred swimming depth nearer tosurface of the test aquaria (during the first exposure days)could be caused by an increase in oxygen consumption ofdaphnids. Various substances are known to change thelocomotion activity of aquatic animals, giving rise to differentphysiological consequences [20, 21, 42]. Other studies haveshown a significant effect of pesticides on swimming motionof Daphnia magna and the magnitude of the change in thefractional dimension was related to the toxic chemical con-centration and the exposure time [23]. Untersteiner et al. [37]described a significant decrease in the average swimmingvelocity of copper-exposed daphnids. Negative phototacticbehavior as an index of chronic copper stress in daphnidswas also found to be elevated [43]. Moreover, in our experi-ments, daphnids first attempted to compensate for higherenergy demands by lowering their swimming depth, to im-prove their oxygen supply and, thereafter, they showed aclear reduction of motility, leading to a sinking out of organ-isms. Therefore, preferred motility receives higher priority inenergy allocation than preferred swimming depth.

Daphnids are common prey items for many planktivores. Forthem, the behavioral properties of their food items (swim-ming speed, horizontal turning, preferred area) are importantcomponents of prey selection, and a reduction of motilitymay dramatically change the prey survival rate. Filter-feed-ing zooplankton must continuously spend energy both forcollection of food and, moreover, for the movement of theirsecond antennae. Their locomotion depends on a constantmuscular activity and is therefore highly energy demanding.For these steadily swimming animals it might be profitable todecrease their locomotive activity under toxic environmentalconditions if the energy input is lower than the overall costsof physiological processes, including movement.

Organic particles and algal cells that settle on the bottomare a very rich source of food for animals equipped to dealwith them. It is possibly for this reason that D. magna oftenoccurs not far above the bottom during the non-exposureperiods. Furthermore, our results show that the decrease inthe mean swimming speed and the sinking to the deeperlayers of the aquarium was caused by a decreased beat fre-quency or power of the second antennae.

We hypothesized that it is possible that inhibited muscularmovement of the second antennae may be caused by theeffects of chemicals on cells at the molecular level, because


there are relations between TBT and increasing intracellularCa2+ levels, decreasing ATP synthesis or phenoloxidase ac-tivities, causing irreversible activation of the sarcoplasmic re-ticulum calcium channel, inhibiting mitochondrial electrontransport, or inhibiting ATPase [38, 44�47]. It is very likelythat both disturbed internal calcium concentrations and in-hibited ATP production could cause pathological effects inDaphnia like immobilization or even death. The inhibition ofmuscle contraction frequency and muscle power could havegreat ecological consequences, especially in situations witha low oxygen level in deeper layers. It appears feasible thatexposed daphnids sink out and suffocate near the bottombefore they can be irreversibly intoxicated by the xenobiot-ics. Even for daphnid predators (such as fish) a phase shiftin swimming behavior due to xenobiotic stress on the preymay have great ecological relevance.

3.2 Reproduction

No mortality occurred in the experiments with female daph-nids under non-exposure or exposure conditions. Newborn

Fig. 3: Reproduction of D. magna(n = 3 groups) exposed to PCB(15 µg L�1), TBT (6.6 µg L�1) orboth (50% each xenobiotic) asmean living juveniles, expressedas difference to the control group.

Reproduktion von D. magna(n = 3 Gruppen) exponiert gegenPCB (15 µg L�1), TBT (6.6 µg L�1)oder eine Mischung beider (50%jeder Substanz). Mittlere Zahllebender Jungtiere als Differenzzur Kontrolle.

Fig. 4: Reproduction of D. magna(n = 3 groups) exposed to PCB(15 µg L�1), TBT (6.6 µg L�1) orboth (50% each xenobiotic) asdead juveniles, expressed asmeans over the day.

Reproduktion von D. magna(n = 3 Gruppen) exponiert gegenPCB (15 µg L�1), TBT (6.6 µg L�1)oder eine Mischung beider (50%jeder Substanz). Tote Jungtiere,dargestellt als tägliche Mittel-werte.


daphnids were, however, found dead in the chemical ex-posed groups; ephippial eggs did not occur.

Reproduction rates were found during the 21 day exposureperiod, as shown in Figure 3. In the controls, (10.6 ± 1.1)living newborn per female and day were noted, expressedas means over the whole experimental time. Methanol expo-sure did not cause significant effects on the daphnids.

The relative reproduction of daphnids, expressed as thechange in the number of living juveniles compared to thecontrol group, is given in Figure 3. There was found no sig-nificant difference in reproduction between treatments andcontrol during the first week. Inhibition of reproductionstarted with the mixture-exposed daphnids on the 9th expo-sure day, followed by TBT exposed animals on the 10th day.

In addition to living newborn daphnids, there were also somedead juveniles found in the TBT and PCB/TBT exposuregroups, in contrast to no mortality in the PCB and controlgroups. These results are illustrated in Figure 4, with signifi-


cant differences between the control and the TBT and PCB/TBT groups. Unfortunately, it was not possible to distinguishbetween daphnids born dead (miscarriage) and those bornliving and died immediately after birth.

Relative reproduction, expressed as the sum of living anddead newborn is given in Figure 5. In the experiments withexposed daphnids, we found only a slight effect of PCB ((9.9± 0.9) living newborn per female and day), but significanteffects of TBT and mixture (Figure 5; expressed as meansover the whole experimental time). A marked inhibitory effect(35.8%) occurred upon TBT exposure ((6.8 ± 176) livingnewborn per female and day). There was no clear reductioncaused by PCB (7%). The xenobiotic mixture had a signifi-cant effect on the number of living newborn (57% decreasedreproduction). The strongest inhibition of reproduction, how-ever, was found with the mixture: the number of living new-born ((4.5 ± 1.5) living newborn per female and day) wassignificantly lower even than that resulting from exposure toTBT. That reduction appears to be more than additive; it isprobably synergistic.

Muscle contraction is not only necessary for horizontal orvertical locomotion but also for movement of the complicatedfilter apparatus (e.g. 2...4 of the trunk limbs, labrium, man-dibles, oesophagus, furkal claw). Daphnids are filter feederand filtration can only be practiced if a particle-bearing cur-rent is drawn or pressed through a filter; this demands a

Fig. 5: Relative reproduction ofD. magna (n = 3 groups) exposedto PCB (15 µg L�1), TBT(6.6 µg L�1) or both (50% eachxenobiotic) compared to controlgroup, expressed as means overthe whole experimental time(significance is marked withasterisk, p = 0.05; U-test). Bothdead (black) and living (hatched)newborn daphnids are shown.

Relative Reproduktion von D.magna (n = 3 Gruppen) exponiertgegen PCB (15 µg L�1), TBT(6.6 µg L�1) oder eine Mischungbeider (50% jeder Substanz) imVergleich zur Kontrollgruppe. Dar-gestellt als Mittelwerte über diegesamte Versuchszeit (*signifi-kant für p = 0.05, U-Test). Darge-stellt sind sowohl tote (schwarz)wie lebende (gestreift) neugebo-rene Daphnien.


rhythmically operating pump [48]. The rate at which daph-nids move water (and food particles) through the carapace isknown to vary widely according to environmental conditions[43]. Therefore, decreased filtration and a resultant reductionin assimilation could lead to lower productive output of theexposed organism, measurable as decreased body growthor reproduction. Here, a significant reduction in the numberof juveniles per TBT exposed female has been observed.Also, a significant reduction in individual body growth (cara-pace length) was found in TBT exposed daphnids (in prep.).Both effects could be caused by a reduction in food intakeand will be investigated in further studies. There are somehints for such an hypothesis; e.g. copper stressed daphnidsalso reduced their filtration rate, body length, and photo-taxis [43].

It is known that daphnids are capable of sexual or parthen-ogenic reproduction and the switch between the two formscan be affected by various environmental conditions, includ-ing chemicals [26]. A NOEC (no-observed-effect concen-tration) of TBT on daphnids was found based on repro-duction: Kühn et al. [49] described the NOEC (21 d) of TBTon daphnids to be 0.16 µg L�1. As is shown for separateexposure to TBT or PCB in our study, a significantly de-creased reproduction rate was seen only in case of TBT(36%), in contrast to the lack of a clear reduction by PCB(7%); also the xenobiotic mixture significantly affected thenumber of newborn (57% decreased reproduction). Here we


found not only a reduced number of living juveniles in TBTand PCB/TBT exposed daphnids, but also the appearanceof dead juveniles in both groups. The sum of living and deadjuveniles was, however, still significantly lower in bothgroups, and it can assumed therefore that mean brood sizewas reduced. In contrast to our work, mean brood size ofdaphnids was a poor index of copper stress [43]. Unfortu-nately, it was not possible in our study to distinguish deadjuveniles from still-born embryos or free swimming young,which died. The developing daphnids could be influencedindirectly by chemicals in the ovaries or directly, when theyare thereafter transferred into the brood chamber [16]. Thereduced mean brood size described in our study couldcaused by indirect effects of chemicals during egg develop-ment in the ovaries leading to abnormalities or even miscar-riages. Additionally, direct effects of TBT or PCB could leadto a reduced survival rate in the brood chamber or in themedium. A similar phenomenon was described by Vietoris[16]; both dead young and rejected eggs were found duringexposure of daphnids to chemicals. In our opinion, a re-duction in the number of young or the occurrence of deadyoung could be indirectly caused, due to chemicals leadingdisturbing the physiology of the mother organism. If the mus-cular contractions of exposed daphnid are inhibited, it canlead to reduced filtration, ingestion and assimilation of food,which is causally related to possible energy investigationsinto reproductive production. Poor environmental conditionsof daphnids can lead to reduced storage of vitellogenin inoocytes [50], and energy transfer from eggs to mother or-ganism (trade off) was even described [51]. Therefore, anincreasing mortality of young is very plausible in chemical-stressed daphnids.

However, bioassays that evaluate only survival and/or repro-ductive responses may underestimate the toxicity of somechemicals; this is particularly likely if the test duration is tooshort to detect significant reductions in life span [43]. ForTBT and PCB, our data show that the 21 day behavioral testwill detect concentrations which significantly reduce repro-duction. In contrast to a clear effect of TBT on the numberof young per female, there was no significant effect of PCBon reproduction. These results could be also attributed tothe higher water solubility of TBT compared to the higherlipophilicity of PCB.

There could be even two different mechanisms influencingthe reproduction of daphnids in our exposure experiments:First, single chemicals are known to be endocrine disruptersand second, there are toxic effects on other physiologicalprocesses (e.g. calcium levels or ATP synthesis) acting atthe same time. The various influences of mixed xenobioticson the organism could be a stress situation that is too greata load for the daphnids’ physiology to cope with.


Although there are various studies investigating the effectsof hormone-mimicking industrial chemicals, the endocrinesystem of invertebrates, including crustaceans, is ratherpoorly understood. Reported hormones in crustaceans are e.g.ecdysteroids, steroids, terpenoids and neuropeptides, whichcontrol processes such as molting, vitellogenesis, repro-duction and sexual differentiation [52]. There are many con-taminants, including PCB and TBT, which could cause endo-crine disruption in crustaceans (e.g. atrazine, cadmium, lin-dane, nonylphenol) with effects such as elevated ecdys-teroid levels, interference with molt, growth, energymetabolism and fecundity, inhibition of larval development,and disrupted testosterone metabolism. In Daphnia magnain particular, different contaminants (Ponasterone A, Cypro-terone acetate, Androstenedione or methoprene) weretested, producing effects such as reduced fecundity, molt-independent growth reduction and reduced offspring num-bers [52]. For instance, TBT may disrupt endocrine functionsin various invertebrates [39, 53]. These findings are similarto the results of Oberdörster et al. [13], who found that theproduction of testosterone metabolites was elevated indaphnids exposed to 1.25 µg L�1 TBT. Therefore, increasinglevels of testosterone caused by TBT exposure can lead toan increased formation of male characteristics. Furthermore,embryo developmental abnormalities associated with sup-pressed ecdyson levels were caused by the antiecdysteroidfungicide fenarimol, which also lead to reduced fecundity ofparental daphnids [27]. Similar mechanisms could be pos-sible in case of TBT, which leads here to a reduced numberof young as described above.

3.3 Toxicity tests and their relevance

Daphnids have become a model system for addressing awide range of ecological and ecotoxicological questions.Nevertheless, it is important to point out that no single testcan yet yield a complete picture of the overall toxic re-sponse, since chemicals may have multiple modes of action[35]. On the other hand, this is an inherent problem in theprediction of acute toxic or chronic effects of waters on or-ganisms, because they are often faced with a mixture ofchemical substances. Therefore, our experiments dealt withthe investigation of the possible additive, antagonistic, orsynergistic effects of xenobiotic mixtures on daphnids. In firstset of experiments, we studied the influence of single sub-stances (TBT or PCB) on daphnid swimming behavior andreproduction and thereafter we tested the hypothesis thatxenobiotic mixtures cause more-than-additive effects onboth parameters.

By investigating quantitative behavioral parameters of ani-mals, it became evident that exposure to single chemicals(TBT: 6.6 µg L�1 or PCB: 15 µg L�1) significantly affectsdaphnids: Exposure led finally to a rapid decrease in mean


swimming activity (PCB: 34%, TBT: 5%) and caused also adecrease in the preferred swimming depth (PCB: 11%, TBT:15%) to nearer the surface. Furthermore, our resultsshowed that the mixture exposure (TBT: 3.3 µg L�1 andPCB: 7.5 µg L�1) altered the quantified swimming behavior(daphnids swam 21% higher and 24% slower) and this ad-verse impact started earlier, compared to exposure to singlechemicals. We concluded that a clear time point dependent,as well as an approximately additive (regarding swimmingdepth and motility), effect of the mixture took place on swim-ming behavior of daphnids.

These findings are in agreement with our own results in pre-vious studies describing the effects of separate exposure toTBT or PCB on ecophysiological parameters of daphnidswith the lowest-observed-effect concentration (LOEC) ofTBT being 0.1 µg L�1 (28d), based on reproduction [inprep.]. The concentration of TBT alone affecting swimmingbehavior and reproduction here was 6.6 µg L�1. These find-ings and those in literature lie in the same range. A TBTconcentration of 2.5 µg L�1 (21 d) was lethal to 60% of theexposed daphnids, as described by Oberdörster et al. [13].Furthermore, they found that lower TBT concentrations elic-ited no adverse effects on molting or reproduction of thedaphnids but the production of hydroxylated, reduced/dehy-drogenated, and glucose-conjugated metabolites of testos-terone were all elevated following exposure to 1.25 µg L�1.These findings indicate that TBT elicits no discernible effectson molting and reproduction of daphnids at sublethal con-centrations, and testosterone metabolism is enhanced atconcentrations approaching those that are lethal to the or-ganism. In addition, the short-time LC50 of organotin ondaphnids was found to be below 14 µg L�1 (24 h; [54]). Theacute toxic TBT level of 2.5 µg L�1 described by Oberdörsteret al. [13] is found to be lower than our concentration usedin reproduction or behavioral experiments. The reason forthis conflict could be the different experimental design. Thedaphnids in these two studies were exposed to TBT underdifferent conditions, with differences in, for instance, the lightcycle (16 h : /8 h or 12 h :12 h), number of animals per beaker(one or 10), beaker used (50 mL, covered with Parafilm or1000 mL, uncovered), age of daphnids (newly released off-spring or 7-day old), food for test animals (daily algal cellsand three times weekly fish food or daily algal cells), fre-quency with which media were changed (every two to threedays or daily). Some or all of these factors will undoubtedlyaffect daphnids and lead to higher or lower effective concen-trations. Also, different salts of TBT yield a wide range of48 h-LC50 values for Daphnia from 2.3 to 70 µg L�1. Mianaet al. [55] calculated the EC50 (immobilization of 50% of theorganisms examined) for daphnids with 12.5 µg L-1 (at 24h)and 9.8 µg l�1 (at 48 h), compared with the NOEC valuesbetween 1.2 µg L�1 and 5.5 µg L�1 (96...24 h); the EC50

level determined using submitochondrial particle (SMP) tests


was as high as 30.2 µg L�1. All these studies describe vari-ous organismal functions upon exposure to TBT under differ-ent experimental conditions, and it is important to comparethe results of the researchers very carefully [56].

This is also true for the investigations of PCB effects ondaphnids and is illustrated by the following study by Nebeker[9]. Continuous-flow and static bioassays were conductedwith survival and reproduction of daphnids as measures ofthe relative toxicity of 8 different PCB’s (Aroclors). It wasfound that Aroclor 1248 was the most toxic to Daphniamagna tested in static tests; the 3-wk LC50 was 25 µg L�1.But Aroclor 1254 was the most toxic PCB to daphnids undercontinuous-flow conditions with a 3-wk LC50 of 1.3 µg L�1.In our own previous studies, the LOEC of PCB was found tobe 1.5 µg L�1 (21 d) when investigating the swimming be-havior [in prep.]. Here, 15 µg L�1 was used successfully inreproduction and behavioral experiments. Additionally,Daphnia pulicaria was exposed to between 50 ng L�1 and10 µg L�1 of a PCB congener (2,2’dichlorobiphenyl) byBridgeham [57], and significant mortality and inhibition of re-production were found at levels as 50...100 ng L�1 in lifetable studies; and no safe level could be determined. In con-trast, Dillon et al. [10] found a lack of PCB congener toxicityon Daphnia magna when they investigated the effects of in-dividual congeners (IUPAC 52, 77, 101, 118, 138, 153, 180)on survival, growth, and reproduction of daphnids. After 21d of static renewal exposure, daphnid survival was very high(up to 100%) in all treatments and unaffected by any PCB,with congeners having little to no detectable sublethal effectson reproduction or growth: the number of neonates pro-duced was either unaffected or even enhanced.

Our hypothesis was that perhaps the inhibited ecophysiolog-ical parameters may be caused by a higher energy demandof other physiological processes such as defense mech-anisms. Biotransformation of toxic compounds in animals in-volves the induction of the biotransformation enzyme sys-tems [38]. In general, biotransformation activity increasesafter exposure to xenobiotics and, therefore, our furtherexperiments deal with possible biotransformation in daph-nids. However, in previous studies we measured a signifi-cantly increased GST activity in gills and liver samples fromfish after exposure to PCB (0.1 µg L�1) or TBT (0.8 µg L�1).Also, other authors described that TBT has been found tointerfere with various enzyme systems. Inhibition effects ofTBT on phase 1 system were described by Oberdörster etal. [13], in addition to the clearly increased activity of GST(phase 2). PCB is known to be a potent inducer of enzymesystems such as the microsomal CYP1A monooxygenasesystem (EROD) and the conjugation enzyme system (cyto-solic GST) in animals [5]. However, the possible biotrans-formation of single or mixed substances could lead to thesituation that the cells and the whole organism could be pro-


tected from physiological and energetic impairment or evendamage and death. The partitioning of energy resources e.g.for biotransformation pathways is one of the most difficulttasks an organism has to solve under the influence ofstress.

From our behavioral observations and ecophysiological re-sults we could not determine whether the observed changesof parameters were symptoms of a partial energy shift, or ifthey were only signs of a global energy saving strategy ofdaphnids, or both. One possible result of destroyed physio-logical homeostasis of daphnids is no doubt the change inoxygen consumption. Therefore, the respiration of exposedanimals should be investigated in further toxicologicalexperiments to calculate the portioning of energy in organ-ism.

Organisms are typically exposed to multiple mixtures of en-vironmental chemicals, but a direct assessment of combinedeffects is not feasible in many cases [18]. Several ap-proaches to the investigation of mixture toxicity have beenused. For instance, effects of chemical mixture of cyanideand trimethyltin were found to be antagonistic in Artemia,contrasting with the synergistic effect of trimethyltin and ber-yllium [12]. It is much more complicated, if one of the chemi-cal consists of different compounds, like Aroclors. For in-stance, the effects of various Aroclors and single PCB con-geners were investigated and described as immunosup-pressive with non-additive (antagonistic) interactions in mice[58]. Models predicting the theoretically expected interactiveeffects of chemical mixtures such as ‘concentration-addition’ and ‘independent action’ are suitable and used bymany researchers [16�18]. This can include the exposureof 100% single substances in various concentrations (e.g.as EC1, EC50), used also in mixture exposures. In contrast,we wanted to know if it is possible to measure synergisticeffects of mixed chemicals using lower concentrations.Therefore, here we used only 50% of each xenobiotic com-ponent for the mixture exposures and as a result we foundeffects of the mixture which were approximately additive(swimming behavior) or even more than additive (repro-duction). Also, DeLong used single doses of PCB or TBT orcombinations of each, when investigating activities of arylhydrocarbon hydroxylase and cytochrome P-4501A in mice[59]. The elevated enzyme activity due to PCB was en-hanced by co-exposure to low levels of TBT, but the highestdoses of TBT inhibited these activities when given in combi-nation with PCB. The induction of cyt P4501A activity in thepresence of both an inducer (PCB) and low concentrationsof an inhibitor (TBT) indicates that TBT does not interferewith the Ah receptor binding, but acts at the transcriptionallevel in rat [60]. However, further studies are needed tounderstand the complete mechanism of interactions of PCBand TBT. Parameters such as swimming behavior and repro-


duction proved to be useful indicators for various effects ondaphnid physiology, caused by PCB, TBT or mixed exposure.

4 Conclusions

The aim of this study was to evaluate how single substancessuch as PCB or TBT influence ecophysiological patterns indaphnids and whether or not the effects of single substanceswould be changed if mixtures of both xenobiotics were used.The results presented demonstrate that effects on swimmingbehavior and reproduction of daphnids were substance-de-pendent and influences of the mixture were found to be e.g.approximately additive (swimming depth, motility) or, mostlikely, approximately synergistic (reproduction). In our tests,single and mixed xenobiotics led to a decreased swimmingspeed and changes in preferred swimming depth in a time-dependent manner. In contrast, daphnid reproduction wasclearly influenced only by TBT and mixed exposures. In ouropinion, all disturbances of the physiological steady state ofanimals can induce adaptive mechanisms and even lead tomore or less pathological effects, which are energy de-manding. The behavioral and physiological changes ob-served are important and sensitive toxicological end-points,showing sublethal disturbances in the organism’s physiologyand allowing discussion of the ecological consequences.

Acknowledgements

The present study was supported by the Deutsche Bundes-stiftung Umwelt, Germany (No. 6000/708).

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[Received: 24 June 2004; accepted: 25 January 2005]

influence of a xenobiotic mixture (pcb and tbt) compared to single substances on swimming behavior...

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