CLONAL VARIATION IN PHYSIOLOGICAL RESPONSES OF DAPHNIA MAGNA TO THE STROBILURIN FUNGICIDE AZOXYSTROBIN

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1 Environmental Toxicology and Chemistry, Vol. 28, No. 2, pp , SETAC Printed in the USA /09 $ CLONAL VARIATION IN PHYSIOLOGICAL RESPONSES OF DAPHNIA MAGNA TO THE STROBILURIN FUNGICIDE AZOXYSTROBIN TRINE PERLT WARMING,* GABI MULDERIJ, and KIRSTEN SEESTERN CHRISTOFFERSEN Freshwater Biological Laboratory, University of Copenhagen, Helsingørsgade 51, DK-3400 Hillerød, Denmark Koeman en Bijkerk Ecological Research and Consultancy, Kerklaan 30, P.O. Box 14, 9750 AA Haren, The Netherlands (Received 17 June 2008; Accepted 14 August 2008) Abstract Because of its high grazing potential, Daphnia magna is an ecologically important species in aquatic food webs. This is especially true in small, shallow ponds lacking fish, where grazing by D. magna may have a relatively higher impact on water clarity as compared to larger lakes. Thus, a reduction in daphnid abundance may have dramatic ecological consequences for shallow ponds. At the same time, shallow ponds in close proximity to agricultural areas likely experience higher concentrations of pesticides because of runoff, spray drift, and drain flow. In the present study, the acute and chronic physiological effects of the strobilurin fungicide azoxystrobin on three clones of D. magna originating from different Danish lakes were evaluated. Significant clonal variation in the sensitivity of D. magna toward azoxystrobin was demonstrated. One clone had a 48-h median lethal concentration (LC50) of mg/l (95% confidence limits [CL], and mg/l), which is comparable to the value widely used in risk assessments (0.259 mg/l). The two remaining clones were far more sensitive, however, and had LC50s of mg/l (95% CL, and mg/l) and mg/l (95% CL, and mg/l), respectively. Furthermore, through respiration measurements and life-table experiments, sublethal stress was shown to exist at exposure to an ecologically relevant concentration (0.026 g/l). Based on these results, we may expect changes in daphnid populations at azoxystrobin concentrations much lower than previously thought. Thus, ponds in the agricultural areas may experience changes in food-web structure even at very low concentrations of azoxystrobin. Keywords Azoxystrobin Fungicide Acute toxicity Chronic toxicity Daphnia magna INTRODUCTION Studies regarding the ecotoxicological effects of toxic substances often are conducted on zooplankton, because they are one of the most sensitive groups of organisms in the aquatic environment. Moreover, they often dominate shallow aquatic ecosystems, especially where fish are not present; thus, adverse effects on zooplankton may have an impact on the ecosystem as a whole. Daphnia is a large-bodied cladoceran zooplankton genus that plays a key role in freshwater pelagic food webs [1] and, therefore, frequently is used in ecotoxicological tests. Azoxystrobin was the first of a novel group of fungicides called strobilurins and became commercially available in 1996, although the first registration of the pesticide in Denmark was in 1998 [2]. Aquatic ecosystems in agricultural landscapes that are treated with pesticides may receive pesticides via runoff, spray drift, and drain flow, and many Danish lakes and ponds have been reported to contain pesticides. In 2001, azoxystrobin was found in Danish surface waters in agricultural areas at concentrations of up to g/l [3]. In Sweden, azoxystrobin have been found in streams at concentrations more than 10-fold higher than those found in Denmark (up to 0.3 g/l in Sweden) [4]. As the use of pesticides in agriculture increases [5], we also may expect the concentrations of pesticides in our aquatic environment to rise. Aquatic ecosystems in Denmark are thus at risk of exposure to azoxystrobin, which may induce adverse effects on nontarget organisms, such as zooplankton. Effects of insecticides and herbicides on zooplankton are * To whom correspondence may be addressed (twperlt@bio.ku.dk). Published on the Web 9/22/2008. well studied (see, e.g., Hanazato [6]), but only a few investigations have focused on the effects of fungicides [7,8]. Recently, the effect of acute exposure to azoxystrobin on Daphnia magna and other aquatic organisms was investigated in relation to synergistic effects with the imidazole fungicide prochloraz, but the effects of azoxystrobin alone were not reported [8]. Furthermore, most often only the median lethal concentration (LC50), median effective concentration, or no-observed-effect concentration using daphnid immobilization as the endpoint are reported, and although these parameters are important tools in many contexts, knowledge regarding the sublethal and/or chronic effects of a given pesticide is vital. Earlier studies have shown that different stressors, such as heavy metals, insecticides, and fungicides, can have sublethal effects on daphnids, such as delayed molting, abnormalities in embryo development, embryonic death, feeding inhibition, and changes in population sex ratios [9 11]. These processes, although not as conspicuous as direct mortality, may still eventually lead to population declines of D. magna, which can have dramatic effects on the ecosystems they inhabit. Information regarding chronic effects of azoxystrobin on daphnids, however, is not available. Another factor that has been studied less thoroughly with respect to effects of pesticides on zooplankton is clonal variation in the sensitivity of D. magna to these substances. Only two studies have shown differential responses of D. magna clones to two toxic substances (cadmium and 3,4-dichloraniline). Both studies showed that clonal differences in acute toxicity tests were more pronounced than those in chronic tests and that the mechanisms of toxicity for a given toxicant may differ under acute and chronic exposure [12,13]. Investigations of clonal variation, however, are more common in studies con- 374

2 Toxicity of azoxystrobin to Daphnia magna Environ. Toxicol. Chem. 28, cerning biotic factors, such as food availability [14], predation [15], or cyanobacterial toxins [16]. Similar to clonal differences in responses to the biotic factors mentioned, clonal differences in responses to the presence of pesticides may influence clonal composition of daphnid populations. Because most or all of the time daphnids reproduce parthenogenetically [17], natural populations normally consist of a few to many clonal genotypes that coexist but fluctuate in response to short-term changes in environmental conditions [18]. Considerable clonal variation in sensitivity toward different toxicants has been shown to exist [12], and although one clone may cope relatively well with one type of toxicant, that clone may well be more sensitive to another. Thus, toxicants, which influence the clonal composition of the population in the direction of reducing clonal diversity, may render the population as a whole more vulnerable to other toxicants. The purpose of the present study was to evaluate the effects of the strobilurin fungicide azoxystrobin on physiological parameters of D. magna. We used acute toxicity tests, life-table experiments, and single-daphnid respiration measurements to describe lethal and sublethal effects of a range of azoxystrobin concentrations. To enable estimation of clonal variation, three clones of D. magna, all isolated from Danish lakes, were tested using each of the three techniques mentioned above. We aimed to examine whether the response of D. magna to azoxystrobin varies significantly between clones and whether clonal variations in the response to the fungicide varied after acute and chronic exposure. Based on the experiments of Baird et al. [12] and Barber et al. [13], we expected a relatively high level of clonal variation in the acute toxicity tests, whereas the lifetable experiments were expected to show subtle effects on reproduction with less distinguishable clonal variation. We also expected to see an indication of sublethal stress expressed as increased rates of oxygen consumption in response to exposure to azoxystrobin. MATERIALS AND METHODS Scenedesmus acutus cultures Batch cultures of the green alga Scenedesmus acutus Meyen (originally obtained from the Max Planck Institute for Limnology, Plön, Germany) were cultured at 19 1 C (mean standard error) in Z8 medium [19] under continuous light from warm-white fluorescent lamps at 25 mol photons/m 2 /s (photon flux density as measured outside the culture vessels). Cultures were harvested during exponential growth by centrifugation for 10 min at 3,100 rpm. The harvested cells were then resuspended in synthetic plankton medium [20] immediately before use as food for the daphnid cultures. The concentration of S. acutus was estimated according to previously established relations between algal biovolume and light extinction (800 nm) [21]. Daphnia magna cultures In all experiments, three clones of the cladoceran species Daphnia magna Straus were used. All the clones were originally isolated from lakes in Denmark: Gammelmosen (GM) in 2003, Herlev Gadekær (HG) in 2005, and Langedam (LD) in The first two were isolated by the Freshwater Biological Laboratory, University of Copenhagen; the last was kindly supplied by O. Kusk (Technical University of Denmark, Lyngby, Copenhagen). All clones have been cultured successfully at the Freshwater Biological Laboratory for several years. The D. magna clones were cultured in synthetic zooplankton medium [20] with S. acutus as the sole food source. Before the experiments, 20 neonates were harvested from well-fed stock cultures and transferred into 500-ml glass jars containing a suspension of 1 mg C/L of S. acutus. The cultures were kept on a plankton wheel ( 1 rpm) at a temperature of 20 1 C, and the room was kept dark to ensure minimal photodegradation of azoxystrobin and to avoid growth of the food alga. The food suspension was renewed every second day. Once the D. magna had been cultured under these conditions for three generations, the animals were defined as acclimated. To minimize maternal effects, only neonates from acclimated mothers were used in the experiments [22]. Neonates for the experiments were taken from the third, fourth, or fifth broods of the third, fourth, fifth, or sixth generations of D. magna. All neonates for a given experiment were taken from a single brood. Acute toxicity tests For each concentration of azoxystrobin (see below), 10 animals per clone were randomly assigned to 25-ml glass vials filled with 25 ml of synthetic zooplankton medium (control) or medium with azoxystrobin (one individual per vial). During the acute toxicity tests, the daphnids were not fed to avoid adsorption of azoxystrobin on food particles. Incubation conditions for the acute toxicity test were as described above. To avoid any risk of oxygen depletion, the medium was exchanged daily. Immobility of the daphnids was recorded after 24 and 48 h of exposure to azoxystrobin. Azoxystrobin from a stock solution (dried azoxystrobin [Sigma-Aldrich, Brøndby, Denmark] dissolved in acetone at a concentration of 1 mg/l) was added to synthetic zooplankton medium. The azoxystrobin concentrations applied were 0.000, 0.005, 0.010, 0.030, 0.060, 0.150, 0.300, 0.600, 1.500, and mg/l, respectively. The same amount of acetone was added to all test solutions, including the control. Previous experiments performed in this laboratory have shown that addition of acetone at concentrations of up to 1 ml/l has no effect on D. magna neonates [23]. Addition of azoxystrobin to synthetic zooplankton medium did not influence the ph of the medium (ph range, ). Life-history experiment The effect of azoxystrobin on life-history characteristics of D. magna feeding on S. acutus was studied for the three D. magna clones. The concentration of azoxystrobin in situ in Denmark can reach levels of up to g/l [23]; therefore, this concentration was used in the life-history experiment. Azoxystrobin from the same stock solution used in the acute toxicity tests was added to synthetic zooplankton medium. Control measurements were conducted in synthetic zooplankton medium with addition of acetone but without azoxystrobin. The final acetone concentration in the medium was 26 l/l ( % of the final volume). It is highly unlikely that the final concentration of acetone could have had adverse affects on D. magna [24,25]. Also, in this part of the present study, the ph of the medium ranged between 7.5 and 7.7, regardless of azoxystrobin. For both treatments, 10 newly released neonates (age, 0 24 h) per clone were sampled randomly from the stock cultures. These neonates were transferred individually into 25-ml glass vials, containing 25 ml of synthetic zooplankton medium (with or without azoxystrobin) and 1 mg C/L of S. acutus. Incubation conditions were the same as those described for culturing D. magna except for medium renewal, which was performed daily. At the start of the ex-

3 376 Environ. Toxicol. Chem. 28, 2009 T.P. Warming et al. periment, the length (defined as the distance from the center of the eye to the base of the tail spine) of all neonates was measured using a stereo microscope (model SD/2PL; Kyowa, Sagamihara, Japan). Growth, reproduction, and mortality were monitored daily for 21 d. The following parameters were investigated during the experiment: Age at first reproduction (release of first clutch into the brood chamber), size of the first clutch, and mean size of all clutches. The dry weight of D. magna was calculated from previously established length/ weight relationships [26]. Inspection for molting and reproduction was carried out daily during renewal of the medium. Respiration experiments Respiration measurements were carried out using a microrespiration system consisting of closed respiration chambers and a calibrated, Clark-type oxygen microsensor (model OX- MR; Unisense A/S, Århus, Denmark). Calibration of the oxygen sensor was performed with zooplankton medium bubbled with air (100% saturation) and a solution of sodium dithionite bubbled with pure nitrogen (0% saturation). Respiration rates were measured in either medium containing g/l of azoxystrobin or in control medium containing 26 l/l of acetone. For each treatment, 10 adult female D. magna were chosen from the stock culture and measured using a stereo microscope. The animals were rinsed three times in pure zooplankton medium to avoid transfer of food particles, and the single daphnids were then transferred to 500- l respiration chambers. The chambers were placed in a waterbath (20 1 C), and the animals were allowed to acclimate in the chambers for 10 min before measurements were performed. The oxygen concentration was measured in each chamber for 15 min, and D. magna respiration rates were calculated from the linear slope of the oxygen concentration plotted against time. Azoxystrobin concentrations All azoxystrobin concentrations given in the present study are nominal concentrations. In an earlier study regarding the effect of azoxystrobin on aquatic invertebrates performed at this laboratory, actual azoxystrobin concentrations were measured and compared to the nominal concentrations using gas chromatography mass spectrometry and liquid chromatography mass spectrometry [23]. The nominal and actual concentrations did not differ significantly in this earlier study, and because identical work procedures were used, the risk of erroneous dosage in the present study was deemed to be minimal. Another potential path to incorrect concentrations of azoxystrobin is degradation of the fungicide in the test medium. Azoxystrobin is a relatively stable compound in aqueous solution, with the major pathways of degradation being biological breakdown and photolysis (information obtained from Sigma-Aldrich). The methodology of the present study was designed to prevent both modes of degradation. All experimental vials as well as stock solutions were kept in darkness to prevent photolysis, and the test medium was renewed daily in both the acute and chronic exposure experiments. The daily renewal ensured that biological breakdown of the fungicide was minimal. Thus, the actual concentrations are unlikely to differ significantly from the nominal ones. Statistical analyses For the acute toxicity tests, the number of immobilized daphnids in each clone was plotted against the azoxystrobin concentration, and a 48-h LC50 as well as 95% confidence limits (CL) were calculated by the standard probit procedure [27]. Differences between the LC50s of the three clones were determined using the principle of nonoverlapping 95% CL [28]. The statistical significance of possible differences between survival functions for the two treatments in the life-history experiment was determined with log-rank tests using Statistica software (1999 edition; StatSoft, Tulsa, OK, USA). Differences in daphnid growth (increase in length) between controls and treatments were analyzed with repeated-measures analysis of variance. Differences in mean clutch size, number of clutches, total number of live juveniles, and age at first reproduction were tested using Student s t tests. Differences in respiration rates between controls and azoxystrobin treatments also were tested using Student s t tests. Acute tests RESULTS The acute toxicity of azoxystrobin, as defined by the 48-h LC50, differed between clones. Two clones, GM and HG, showed similar responses to the toxin (LC50: GM, mg/l [95% CL, and mg/l]; HG, mg/l [95% CL, and mg/l]), but the LD clone had a significantly (nonoverlapping 95% CL) higher 48-h LC50 (0.277 mg/l [95% CL, and mg/l]), indicating that this clone was less sensitive to azoxystrobin compared with the other two. The average LC50 for all three clones was mg/l (standard error, mg/l). Figure 1 shows the survival of the three clones after 48 h of exposure to azoxystrobin. Life-table results Continuous exposure of daphnids throughout their life cycle (21 d) to an azoxystrobin level comparable to the highest value measured in Danish streams (0.026 g/l) had no significant effect on the survival of any of the clones (data not shown, all p values 0.08); thus, clonal variation could not be determined. Mortality never exceeded 20%. Sublethal effects Daphnid growth was not affected by continuous exposure to g/l of azoxystrobin throughout the life cycle, as the repeated-measures analysis of variance yielded no significant effect on any clone (all p values 0.161) (Fig. 2). Reproduction Responses in daphnid reproduction parameters to chronic exposure to azoxystrobin (0.026 g/l) differed between the three clones (Fig. 3). The GM clone showed no significant differences between the azoxystrobin treatment and the control in any of the measured endpoints, but the number of clutches released, mean clutch size, and total number of live juveniles produced were all lower in the azoxystrobin treatment than in the control. In contrast, age at first reproduction was higher in the azoxystrobin treatment than in the control. For HG and LD clones, however, the picture was reversed. These clones released their first broods significantly earlier in the azoxystrobin treatments compared to the control. The LD clone also had significantly higher total numbers of juveniles, and the clutches were significantly larger in the azoxystrobin treatment than in the control.

4 Toxicity of azoxystrobin to Daphnia magna Environ. Toxicol. Chem. 28, Fig. 1. Survival of the three clones of Daphnia magna (GM Gammelmosen; HG Herlev Gadekær; LD Langedam) after a 48-h exposure to the strobilurin fungicide azoxystrobin. Respiration The specific respiration rate of D. magna increased significantly, by 38 to 43%, for all three clones (Student s t test, all p values 0.03) following exposure to g/l of azoxystrobin. Moreover, the HG clone had significantly lower specific oxygen consumption rates than the GM and LD clones (Student s t test, all p values 0.001) (Fig. 4). DISCUSSION Generally, the HG and GM clones exhibited similar responses to the varying modes of exposure to azoxystrobin, whereas the LD clone tended to separate from the others. The LD clone, which was the least sensitive clone in the acute test, was the only one to show significant stress symptoms in three of the four measured reproductive parameters in the life-table experiment. However, whereas the HG and GM clones were more sensitive than the LD clone to azoxystrobin in the acute toxicity tests, they were less sensitive in chronic tests. This is in accordance with the findings of Hietala et al. [16], who found a similar inverse response of various clones in acute and chronic experiments with D. magna exposed to toxic cyanobacteria. Also, in a series of studies, Barber, Baird, and coworkers obtained similar patterns (i.e., less clonal variation in chronic as compared to acute studies) in the clonal variation of D. magna exposed to two toxicants (cadmium and 3,4- dichloroaniline, a metabolite of the herbicide propanil) [12,13]. The clonal variation in responses may indicate that the clones have different methods to cope with azoxystrobin stress. As mentioned, the acute sensitivity of D. magna exposed to azoxystrobin varied significantly between clones, with the GM and HG clones being the most sensitive and the LD clone being more than half as sensitive. In other studies, the 48-h LC50s determined for D. magna varied between 0.26 and 0.53 mg/l [23,29,30]. These values are considerably higher than the mean 48-h LC50 for all three clones found in the present study ( mg/l) but are similar to the value found for the LD clone (0.277 mg/l [95% CL, and mg/l]). This means that these studies used strains of D. magna that had a sensitivity to azoxystrobin similar to that of our LD clone. It is interesting to note that the LD clone has been kept in laboratory culture for much longer (since 1978) than the GM and HG clones (since 2003 and 2005, respectively; the present study was carried out in ). Thus, the age of the cultures may play a role in the susceptibility of clones to various toxicants (see also Muyssen et al. [31]). This point is important in that natural populations may be more or less vulnerable than laboratory cultures, which implies that laboratory experiments may be ill-suited for extrapolation to natural conditions. In earlier work from this laboratory, a natural clone of Daphnia galeata was approximately twofold less sensitive to azoxystrobin than a laboratory strain [23], which means that further studies comparing the susceptibilities of laboratory test animals and wild populations to pesticides are needed. So far, risk assessments of spray drift, runoff, and so on have been based on D. magna LC50s of mg/l [29,30]. Two of the three clones used in the present experiment, however, had LC50s that were lower than 0.1 mg/l, which means that the values cited in the literature probably are too high to support a reliable risk assessment. In the future, studies with more than one clone of D. magna, and studies on natural populations as well as laboratory cultures, would be prudent when risk assessments are being made. The life-table experiments showed that neither growth nor survival of D. magna was affected by chronic exposure to a low concentration (0.026 g/l) of azoxystrobin, but more subtle effects were observed in the reproductive behavior of the daphnids. Compared to the controls, the azoxystrobin-treated HG and LD clones reproduced significantly earlier, and the azoxystrobin-treated LD clone gave birth to significantly higher numbers of juveniles in significantly larger clutches. Early reproduction as well as increased clutch size are common reactions to varying kinds of sublethal stress in daphnids [32]. All three D. magna clones showed strongly increased respiration rates (38 43%) in response to exposure to very low concentrations of azoxystrobin. An increase in respiratory demand is of great ecological importance, because it will reduce energetic reserves for growth and reproduction, thereby lowering the overall fitness of the daphnids. The reason that no

5 378 Environ. Toxicol. Chem. 28, 2009 T.P. Warming et al. Fig. 2. Growth curves for the three clones of Daphnia magna (GM Gammelmosen; HG Herlev Gadekær; LD Langedam) exposed to g/l of azoxystrobin for 21 d. Data points represent averages of 10 replicates. Error bars indicate standard errors. effect was observed on growth in the chronic exposure experiment in the present study likely is that the daphnids had an ample food source, which was renewed daily. Thus, their increased metabolic demand was met by the available food. Consequently, under environmental conditions, the increase in metabolic demand caused by exposure to azoxystrobin is likely to have the highest detrimental effect on daphnid populations in situations where low food availability limits growth. Daphnid respiration rates have been measured numerous times in the past. Most of the earlier studies have reported values comparable to the rates obtained in our experiments (2 8 l O 2 /mg dry wt/h) [33 37], whereas one study has reported values that are higher by a factor of approximately 100 ( 448 lo 2 /mg dry wt/h) [38]. The HG clone had significantly lower specific respiration rates than the other two clones, but the measured rates ( lo 2 /mg dry wt/h) are still well within the range of the previously published values. A possible explanation may be a difference in swimming behavior between the HG clone and the other clones. A change in daphnid swimming behavior also could be

6 Toxicity of azoxystrobin to Daphnia magna Environ. Toxicol. Chem. 28, Fig. 3. Reproduction parameters of the three clones of Daphnia magna (GM Gammelmosen; HG Herlev Gadekær; LD Langedam) following exposure to g/l of azoxystrobin (gray bars) or control (white bars). Asterisks indicate significant differences ( p 0.05). All values are averages of 10 replicates. Error bars indicate standard errors. The HG clone daphnids in the control treatment all released their first clutch on the same day; thus, the standard error on this value is zero. argued as a possible explanation for the observed increase in respiration rates following exposure to azoxystrobin, but it has been shown previously that exposure to azoxystrobin decreased rather than increased D. magna swimming velocity by 14% [23]. Also, the frequency of heart rate, thoracic limb beating, and mandible and postabdominal claw movements all have been shown to decrease following exposure to azoxystrobin [23]. Thus, it is evident from the results obtained through lifetable and respiration experiments in the present study that exposure to azoxystrobin, even at very low concentrations, induces stress and increased metabolic cost to D. magna, which will result in reduced fitness and may lead to population decrease. Three clones were tested, and each one turned out to be significantly different in sensitivity to azoxystrobin. Because D. magna often is a key species in ponds and small lakes, this may have visible effects on the ecosystems as a Fig. 4. Specific respiration rates of the three Daphnia magna clones (GM Gammelmosen; HG Herlev Gadekær; LD Langedam) exposed to g/l of azoxystrobin (gray bars) or control (white bars). All values are averages of 10 replicates. Error bars indicate standard errors. whole; for example, reduced grazing pressure on the phytoplankton may lead to decreased water transparency and a subsequent deterioration of macrophyte flora. The present results indicate potential impacts on natural cladoceran populations at realistic surface water concentrations of azoxystrobin and clonal variation in sensitivity toward the fungicide in D. magna. Therefore, further tests, using more natural test conditions and natural, field-collected populations of daphnids, are needed to assess the ecological consequences of exposure to azoxystrobin. Acknowledgement Jette V. Sandsted (Freshwater Biological Laboratory) is acknowledged for technical assistance, O. Kusk (Technical University of Denmark) for providing a culture of the D. magna (LD) clone, and Unisense (Århus, Denmark) for providing the Micro Respiration setup. REFERENCES 1. Lampert W, Sommer U Limnoecology. New York, NY, USA. 2. Jørgensen LN, Nielsen GC Reduced dosages of strobilurins for disease management in winter wheat. Proceedings, Brighton Crop Protection Conference, Pest and Disease, Brighton, UK, November 16 19, 1998, pp Århus Amt Forekomst af pesticider i vandhuller i Århus Amt. Technical Report. Århus Amt, Natur og Miljøkontoret, Århus, Denmark. 4. Ulén B, Kreuger J, Sundin P Undersökning av bekæmpningsmedel i vatten från jordbruk och samhälle år Rapport 2002:4. Ekohydrologi 63: Danish Environmental Protection Agency Bekæmpelsesmiddelstatistik Technical Report. Orientering fra Miljøstyrelsen, Copenhagen, Denmark. 6. Hanazato T Pesticide effects on freshwater zooplankton: an ecological perspective. Environ Pollut 112: Van den Brink PJ, Hattink J, Bransen F, Van Donk E, Brock TCM Impact of the fungicide carbendazim in freshwater microcosms. II. Zooplankton, primary producers, and final conclusions. Aquat Toxicol 48: Cedergreen N, Kamper A, Streibig JC Is prochloraz a potent synergist across aquatic species? A study on bacteria, daphnia, algae, and higher plants. Aquat Toxicol 78: Kast-Hutcheson K, Rider CV, LeBlanc GA The fungicide

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