Multicomponent molluscicide mixtures for zebra mussel control

Journal of Great Lakes Research 38 (2012) 317–325

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Multicomponent molluscicide mixtures for zebra mussel control Raquel Costa a, b,⁎, 1, 2, David C. Aldridge c, 3, Geoff D. Moggridge a, 1 a b c

Department of Chemical Engineering and Biotechnology, University of Cambridge, New Museums Site, Cambridge CB2 3RA, UK Department of Chemical Engineering, University of Coimbra, Pólo II, Rua Sílvio Lima, 3030-790 Coimbra, Portugal Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK

a r t i c l e

i n f o

Article history: Received 21 September 2011 Accepted 14 March 2012 Available online 17 April 2012 Communicated by Paul Helm Keywords: Biofouling Dreissena polymorpha Pallas Invasive bivalves Mixture toxicity Pest control

a b s t r a c t Intuitively, it is reasonable to expect enhanced control of the biofouling zebra mussel through multicomponent molluscicide cocktails. In this study, the potential of combined potassium chloride, polyDADMAC, niclosamide ethanolamine salt and 2-(thiocyanomethylthio)benzothiazole (TCMTB) for zebra mussel mitigation was investigated. A series of mixtures of varying compositions was tested. First, the combination was considered in its entirety, and the nature of the biocides' joint toxicity was elucidated by adopting a structured classification system previously defined. Then, a central composite experimental design was employed to detail the contribution of each ingredient to the blend performance and ultimately derive an empirical model of mixture effects to optimise the formulation composition. Whilst the action of some of the toxins was synergised, the blend does not appear promising for zebra mussel control. Overall, the chemicals acted less than additively and, under some circumstances, antagonistic effects were observed. Although these results do not immediately lead to a new approach to pest mitigation, the study highlights aspects that are of practical relevance for the design of combined chemical treatments. In particular, this work recalls the funnel hypothesis from the field of ecotoxicology (blends tend to be additive as the number of ingredients increases), which may provide key guidance in the mixture design process. Furthermore, the study shows that multiple biocides do not necessarily ensure improved zebra mussel mitigation, and therefore the nature of their combined effects should always be carefully examined. The systematic procedure proposed here to critically design biocide blends is useful in this context. © 2012 International Association for Great Lakes Research. Published by Elsevier B.V. All rights reserved.

Introduction The freshwater mussel Dreissena polymorpha Pallas, commonly known as the zebra mussel, is one of the world's most economically and ecologically important pests. The species, native to the basins of the Black and Caspian Seas, has remarkable dispersal potential and an ability to colonise a variety of environments and habitats (Claudi and Mackie, 1994; McMahon, 1996). It is well established in many waterbodies in Western Europe and North America, and it continues to spread (Minchin et al., 2002; Pimentel et al., 2005). In addition to the negative effects on the infested ecosystems, the zebra mussel has a major impact on freshwater-dependent industries. Drinking water treatment facilities and process cooling systems are especially vulnerable to the species' biofouling activity (Claudi and Mackie, 1994; Elliott et al., 2005). ⁎ Corresponding author. Permanent address at: Department of Chemical Engineering, University of Coimbra, Pólo II, Rua Sílvio Lima, 3030-790 Coimbra, Portugal. Tel.: + 351 239798700; fax: + 351 239798703. E-mail addresses: [email protected] (R. Costa), [email protected] (D.C. Aldridge), [email protected] (G.D. Moggridge). 1 Tel.: + 44 1223 334777; fax: + 44 1223 334796. 2 Tel.: + 351 239798700; fax: + 351239798703. 3 Tel.: + 44 1223 33660; fax: + 44 1223 336676.

The application of lethal toxicants or substances that impair the ability of the animals to attach to hard surfaces is the foremost strategy in most industrial zebra mussel control programmes (Claudi and Mackie, 1994; Post et al., 2000; Mackie and Claudi, 2010). Compared to other control strategies, such as the use of filters, the physical cleansing of infested structures or the coating of vulnerable surfaces with antifouling materials, a chemical control approach tends to be more cost-effective and versatile. It may be implemented in existing facilities without major structural changes and it provides full protection of the system against a range of biofouling agents (Post et al., 2000). In spite of these advantages, a chemical control method raises concerns related to the selectivity and cost of some toxins. Due to these concerns, the need to discover new molluscicides and/or develop improved application strategies has been acknowledged (e.g. Aldridge et al., 2006; Costa et al., 2008). Combining biocides to take advantage of their cumulative and synergistic effects intuitively appears as a promising avenue to explore in this context. Although this strategy has proved successful for the mitigation of several terrestrial and aquatic pests (Ahmad, 2004; Singh et al., 2005), it has not been comprehensively investigated for the case of invasive bivalves (Costa et al., 2011). For the zebra mussels, only a limited number of mainly binary molluscicide mixtures have been examined, and generally the matter has been approached from an empirical rather than

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R. Costa et al. / Journal of Great Lakes Research 38 (2012) 317–325

systematic perspective (van Benschoten et al., 1992; Elzinga and Butzlaff, 1994; Wildridge et al., 1998a; Mackie and Claudi, 2010). In a recent work, Costa et al. (2011) evaluated the potential of a binary mixture of poly(diallyldimethyl ammonium chloride) (polyDADMAC) and potassium chloride for zebra mussel mitigation based on the systematic characterisation of the nature of the biocides' joint effects. A structured approach to perform such characterisation, derived from the ecotoxicology background on mixture toxicity, was proposed in that work. It is reasonable to expect increased lethal effects, and hence enhanced control, when multicomponent rather than binary mixtures are applied. In the present study, the potential of mixtures of potassium chloride, polyDADMAC, niclosamide ethanolamine salt and 2-(thiocyanomethylthio)benzothiazole (TCMTB) for zebra mussel mitigation is discussed. The systematic investigation approach previously proposed for binary mixtures (Costa et al., 2011) is extended to the analysis of the performance of multicomponent molluscicide blends. The quaternary combination above was selected for examination on the following grounds. From the ecotoxicological experimental evidence on mixture toxicity it appears that there is a propensity for blends to be additive as the number of components increase (Warne and Hawker, 1995; Warne, 2002) whilst more than additive combinations are more beneficial from the control perspective (Costa et al., 2011). Furthermore, the possible increased effectiveness of multiple chemicals has to be balanced with potential operational and effluent purification issues related to the dosage of a large number of substances into the system. For these reasons, mixtures of a moderate number of molluscicides are most likely to be preferred in practice, and hence a fairly simple mixture of four biocides was chosen for investigation. The blend incorporates potassium ions (dosed as potassium chloride) and polyDADMAC, whose joint toxic effects on the zebra mussel have previously been shown to be more than additive (Costa et al., 2011). To these chemicals, niclosamide ions (applied as niclosamide ethanolamine salt) and TCMTB have been added. These chemicals are well-accepted zebra mussel control agents (McMahon et al., 1993; Waller et al., 1993; Fisher et al., 1994; Wildridge et al., 1998b; Sprecher and Getsinger, 2000) and act through different mechanisms, which maximises the chances of exerting more than additive effects. Potassium ions, polyDADMAC and TCMTB are believed to cause zebra mussels' death by disrupting gas exchange in the gills by either provoking the depolarisation of (Fisher et al., 1991; Durand-Hoffman, 1995), adsorbing to (Gloxhuber, 1974; Post et al., 1996), or being generally corrosive to (McMahon et al., 1993; Nikl and Farrell, 1993; Walsh and O'Halloran, 1997; Sprecher and Getsinger, 2000) the cell membranes, respectively. Niclosamide ions exert their toxic action by interfering with the cellular respiratory processes (Ishak et al., 1970; Andrews et al., 1982; Mallatt et al., 1994). Systematic evaluation of biocide combinations for zebra mussel control The study of the effects of chemical mixtures on aquatic biota is well-established in ecotoxicology (e.g. Backhaus et al., 2003; Svendsen et al., 2010). Although the investigation of mixture toxicity in the ecotoxicological and pest control contexts involves different issues, the fundamental questions being addressed in both fields are similar, and tools from the former may be of great use in the design of toxin cocktails for the mitigation of aquatic nuisances. However, the links between these two areas of research have not been thoroughly explored. In short, the previous approach to systematically evaluate the potential of binary biocide combinations for zebra mussel control (Costa et al., 2011) involves a two-level classification scheme, and it uses the concentration addition concept (Sprague, 1970; Könemann and Pieters, 1996; Faust et al., 2001; de Zwart and Posthuma, 2005) as a reference response. In the present paper, this structured evaluation method is extended to the design of complex molluscicide mixtures.

When referring to multiple chemicals, the concentration addition concept implies that all toxicants are non-interactive and share a common mode of action, being biologically equivalent at the target site. This mechanistic model is mathematically expressed by (Könemann and Pieters, 1996; Backhaus et al., 2000; Faust et al., 2001; de Zwart and Posthuma, 2005): n n X X cx;i ¼ TU x;i ¼ TU x;mix ¼ 1 LC x;i i¼1 i¼1

ð1Þ

where n denotes the number of chemicals in the mixture; i (i = 1,…, n) refers to a generic substance; cx,i is the concentration of the compound i in the mixture that elicits a lethal effect of magnitude x; LCx,i is the c equivalent effect concentration of component i; the quotient TU x;i ¼ LCx;i x;i is the number of toxic units of the ingredient i in the mixture; and TUx, mix is the total number of toxic units in the blend. Similar to binary blends, the first level of the joint toxicity classification system for multicomponent mixtures is used to refer to a specific biocide i of particular interest and judge whether its toxic action is synergised (TUx,i below one), antagonised (TUx,i above one) or not affected (TUx,i equals one) in the presence of the remaining chemicals in the mixture. However, the concepts of antagonism, synergism and inexistence of joint effect cannot be strictly applied to a multicomponent mixture as a whole. In such blends, different components may exhibit different variations of their individual toxicities, and the overall toxicity of the mixture reflects the balance between all these variations. At the second level of the joint toxicity classification system, mixtures where synergism occurs are treated as a whole and their performance is judged based on the overall toxic load necessary to elicit the lethal response under consideration. The categories used in the analysis of binary mixtures – additive (TUx,mix equals one), more than additive (TUx,mix below one) or less than additive (TUx,mix above one) – can be directly extended to the characterisation of complex mixtures. However, the convenient graphical representation of TUx,mix in a isobologram is not feasible for blends with more than three components. The ultimate assessment of the potential of possible multicomponent molluscicide blends for pest control is performed by employing similar criteria as in the case of binary mixtures (Costa et al., 2011). The selection of concentration addition as a reference response in the proposed classification system does not imply that all molluscicide blends are expected to comply with this model, which mechanistically refers to non-interactive chemicals with similar modes of action (Plackett and Hewlett, 1952; Könemann and Pieters, 1996). Instead, this reference response was chosen for convenience (Costa et al., 2011). Whilst the analysis of the toxicity of binary mixtures is facilitated by their simplicity, in the case of multicomponent combinations the complexity of the experimental procedures required to examine in detail the chemicals' joint effect increases with the number of ingredients. Such complexity is one of the reasons why the analysis of combined toxicity has been typically reduced to the testing of equitoxic formulations, following fixed ratio designs (e.g. Faust et al., 2001; Warne, 2002). Equitoxic blends contain all ingredients at the same fraction of the respective individual toxicity (e.g. median lethal concentration or no observed effect concentration). In fixed ratio study designs, a series of dilutions of a given formulation with varying concentrations but constant relative proportions of the constituents is tested to experimentally describe the dose–response relationship of the formulation. The main drawback of integrative study approaches involving equitoxic formulations and fixed ratio designs, where the mixture is treated as a whole, is the fact that they provide limited information on the individual contribution of each chemical to the mixture toxicity, which is important for the design of pest control strategies. The systematic variation of the mixture composition and the testing of non-

R. Costa et al. / Journal of Great Lakes Research 38 (2012) 317–325

equitoxic blends provide a fuller account of the toxicants' combined action. For complex mixtures, this is more effectively achieved through statistical experimental designs, integrated with multivariate data analysis techniques. Full factorial, fractional factorial and central composite designs as well as response surface, principal components and partial least squares regression analysis techniques may be employed (see, for example, Eide, 1996; Groten et al., 1996; Schoen, 1996; Smith, 1998; and more recently, Groten et al., 2001; Lock and Janssen, 2002; Tajima et al., 2002). The choice of the approach to examine the joint toxicity of multiple chemicals largely depends on the number of components in the mixture and the extent to which the nature of the combined action needs to be characterised. In the present study, the ecotoxicology-inspired view on mixture toxicity discussed in this section was applied to evaluate the potential of mixtures of potassium chloride, polyDADMAC, niclosamide ethanolamine salt and TCMTB for zebra mussel control. Materials and methods Experimental design: three-phase study In the first phase of the study, dose–response data for the individual toxins were obtained. These were used as reference data for mixture toxicity analysis. In the second phase, the combination was considered in its entirety, and the joint toxicity of the four biocides was determined by following a fixed ratio design. Because the nature of the joint effects of the chemicals could depend on the blend's composition (Warne, 2002), two different formulations were examined. In the first (called LC50 formulation), the chemicals were mixed in the relative proportion of their median lethal concentrations. In the second formulation (designated as LC10 formulation), the toxins were present at the ratio of their individual lethal concentrations for percentile 10. The dose–response data obtained in this set of experiments was analysed based on the combined action classification system presented in the previous section. After getting a general appreciation of the potential of the quaternary combination for zebra mussel control, the aim of the third phase of the study was to examine the contribution of each ingredient to the blend performance, deriving an empirical model of mixture effects. This type of data is essential from the practical application standpoint because it allows the formulation to be optimised. A series of combinations were tested in this phase. The test treatments followed a central composite experimental design and response surface analysis was employed (Box and Draper, 1987; Montgomery and Runger, 1999). Test organisms In mid-summer (July), adult zebra mussels were collected from the walls of a filter bed in a water treatment plant in London (UK) by using a paint stripper blade to carefully cut their byssus threads. Immediately after collection, the animals were transported to the laboratory in field water, and individuals with shell length in the range 20 to 30 mm were selected and thoroughly rinsed. The mean shell length of the specimens used in the study was 25.9 ± SE 0.8 mm. The test organisms were held in aerated dechlorinated municipal water in a temperature-controlled chamber at 18 °C (±1 °C), on a 12 h-dark/12 h-light cycle. The toxicity tests, which were conducted in the temperature-controlled chamber under the holding conditions, were initiated within 1 week of collection. Dechlorinated municipal water was used in all experiments. Single biocide toxicity tests Sets of ten mussels were placed into 75 1-litre containers holding 500 ml of continuously aerated water. The animals acclimated for 48 h. Those that did not attach to the bottom of the containers within

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this period (no more than 10% in all vessels) were discarded. As a result, at the moment of dosing, three out of the 75 test pots contained nine rather 10 mussels, which was taken into account in the mortality data analysis. The toxins were dosed so that the specimens were exposed to the following individual treatments: (i) 25, 50, 100, 200, 400 and 800 mg/l of potassium chloride, applied as laboratory grade reagent with purity above 99% in weight (Fisher Scientific UK Ltd, Loughborough, UK); (ii) 10, 20, 40, 120, 360 and 1080 mg/l of polyDADMAC, dosed as a proprietary product containing more than 85% in weight of polymer (SNF UK Ltd, Normanton, UK); (iii) 0.048, 0.096, 0.192, 0.250, 0.324 and 0.422 mg/l of niclosamide ethanolamine salt, applied as Bayluscide® WP 70, which contains 83.1% in weight of the toxin (Bayer AG, Leverkusen, Germany); and (iv) 10, 20, 40, 120, 360 and 1080 mg/l of TCMTB, dosed as BULAB® 6009, whose weight fraction of active ingredient is 30% (Buckman Laboratories International, Inc., Memphis, US). The four chemicals were applied individually by following the dosing scheme intended for the mixture bioassays. Potassium chloride was applied for 48 h. In the case of the remaining toxins, the treatments lasted for 36 h, initiating 12 h after potassium chloride application. In the control treatment, organisms were not exposed to toxins. Each of the 25 treatments was applied in triplicate, the three replications being randomly distributed in the temperature-controlled chamber. Mortality was monitored 24 h and 48 h after potassium chloride dosing, which corresponded to treatment durations of 12 h and 36 h in the case of the other biocides. Failure to respond to an external tactile stimulus provoked by a blunt probe was used as the death criterion. Dead specimens were discarded at each mortality assessment. The results of the bioassays were analysed in terms of the dose–response data obtained at the end of the treatments. Integral mixture toxicity tests The individual median lethal concentrations and lethal concentrations for percentile 10 after the desired exposure periods, obtained from the single biocide toxicity tests, were used to define LC50 formulation and LC10 formulation. The proportions in which the four chemicals were mixed in the two formulations are presented in Table 1. The two quaternary formulations were tested by following a procedure similar to that implemented in the single biocide toxicity tests. Each formulation was tested at 11 different overall concentrations (cmix): 0.75, 1.5, 3, 6, 12, 24, 48, 96, 192, 384 and 768 mg/l. The specimens were pre-treated with potassium chloride for 12 h and then exposed to the four chemicals for 36 h, which resulted in 48-hour overall treatments. The pre-exposure of the test organisms to potassium chloride potentially allowed for the salt to promote the uptake of the other toxins (Costa et al., 2011). In the control treatment, organisms were not exposed to toxins. Each of the 23 treatments was applied in triplicate. The mortality in the test containers was assessed 24 h and 48 h after the commencement of the treatments. The results of the bioassays were analysed in terms of the mortality data obtained at the end of the treatments. Central composite design toxicity tests Combinations of the biocides were tested following a central composite design, consisting of 27 experimental points: 16 cube points, 8 star points and 3 centre points (Box and Draper, 1987; Montgomery and Runger, 1999). Each toxin was tested at five linearly equidistant concentrations, coded as −2, − 1, 0, 1 and 2 (Table 2). These concentrations were chosen so that the centre points corresponded to one-fourth of the median lethal concentrations of the individual biocides after the desired exposure period, and the lowest dosage levels corresponded to the absence of toxins. By following this approach, the four toxins were tested at similar proportions of their individual toxicities. At the central dosage levels (0), the chemicals

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Table 1 Composition of the two formulations tested in the integral mixture toxicity study. The compositions are expressed in terms of the weight fraction of the generic biocide i, pi, given by the quotient between the concentration of the component in the formulation (ci) and the overall mixture concentration (cmix). Biocide

Weight fraction of the biocide in the formulation LC

LC

ci 50;i ¼P LC50 formulation pi ¼ cmix 4

ci 10;i LC10 formulation pi ¼ cmix ¼P 4

LC 50;i

LC 10;i

i¼1

Potassium chloride PolyDADMAC Niclosamide ethanolamine salt TCMTB

i¼1

0.447 0.111 3.21 × 10− 4 0.441

0.747 0.0434 8.24 × 10− 4 0.208

Table 2 Codification of the biocide concentrations tested in the central composite experimental design. Biocide

Potassium chloride PolyDADMAC Niclosamide ethanolamine salt TCMTB

Concentration associated with each code (mg/l) −2

−1

0

1

2

0 0 0 0

30.9 7.67 2.21 × 10− 2 30.4

61.8 15.4 4.42 × 10− 2 60.9

92.6 23.0 6.64 × 10− 2 91.3

124 30.7 8.85 × 10− 2 122

were applied at the same fraction of their single toxicity. At the dosages levels −1, 1 and 2 this was not exactly the case, because their individual dose–response relationships are not parallel (Fig. 1). However, the difference between the respective individual toxicity proportions at levels −1, 1 and 2 was not significant. Each experimental point was tested in triplicate. In general, the toxicity tests were conducted as described above. The test organisms were pre-treated with potassium chloride for 12 h before they were exposed to the combinations containing this salt. In three control vessels, mussels were not exposed to toxins. The overall treatments lasted for 48 h, corresponding to an equivalent exposure to potassium chloride and to treatment durations of 36 h in the case of the other biocides. The mortality in the test containers was assessed 24 h and 48 h after the commencement of the treatments. The results of the central composite design toxicity tests were analysed in terms of the responses produced at the end of the treatments.

Central composite design and response surface data analysis A second-order response surface model (Box and Draper, 1987; Montgomery and Runger, 1999) was fitted to the data obtained in the central composite design toxicity tests. In the regression analysis, arcsine transformed mortality percent values (Zar, 1999) were used. Statistical analyses The statistical methods for general data analysis were used as outlined by Zar (1999) and implemented in STATISTICA (StatSoft, Inc. (2003), STATISTICA (data analysis software system), version 6. www. statsoft.com). Dose–response data were modelled by Probit analysis using the software StatsDirect (StatsDirect, Ltd. (1990), StatsDirect statistical software. www.statsdirect.com). A significance level of 5% was used. Results Single biocide toxicity tests

Data analysis Systematic description of the nature of the biocides' joint action The results of the integral mixture toxicity tests were analysed by employing the proposed structured classification system in order to elucidate the nature of the combined action of the four biocides. The analysis was performed for LC50 formulation and LC10 formulation separately. Lethal responses in the range of 10 to 90% were considered. The magnitude of the response (x) was first specified. Then, the equivalent lethal concentrations of the individual toxins (LCx,i) were determined from the results of the single biocide toxicity tests by Probit analysis. The composition of the experimental combined treatments was converted into a toxic unit scale by dividing the concentration of each component (ci), related to the overall mixture concentration (cmix) (as detailed in Table 1 and originally expressed in mg/l), by the respective equivalent effect concentration (see Eq. 1 for details). The number of toxic units of the mixture able to elicit the lethal response x (TUx, mix) was then determined by Probit analysis. The nature of the chemical's joint action was further examined by analysing the number of toxic units of the individual components in the mixtures able to cause the lethal effect x (TUx,i), which was determined by p ⋅LC the quotient i LC x;ix;mix , where the overall mixture concentration producing the effect x (LCx,mix) was estimated from the results of the bioassays by Probit analysis.

No mortality occurred under the control conditions. The dose–response relationships obtained for potassium chloride, polyDADMAC, niclosamide ethanolamine salt and TCMTB are shown in Fig. 1. Table 3 presents the chemicals' median lethal concentrations and lethal concentrations for percentile 10 after the end of the single biocide exposures, which were involved in the formulation of the treatments assessed in the integral mixture and central composite design toxicity tests. Integral mixture toxicity tests Full survival was observed in the control containers. The mortality produced by the combined treatments was significantly related to the overall mixture concentration (two-factor ANOVA following arcsine transformation of the mortality percent data; F = 73.650; df = 10; p b 0.001; Fig. 2). No significant differences were observed between the effects elicited by LC50 formulation and LC10 formulation (two-factor ANOVA following arcsine transformation of the mortality percent data; F = 0.189; df = 1; p = 0.666). The nature of the biocides' joint action was explored by employing the systematic approach described above. Both LC50 formulation and LC10 formulation were observed to exert less than additive effects over the entire mortality range (Fig. 3). TUx,mix values depended on the response under consideration (two-factor ANOVA; F = 9.658;

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variation of the toxicity of the individual chemicals as they were combined. Fig. 4a shows that, in the case of LC50 formulation, TUx,i were generally lower than one at any response level. The only exceptions occurred in the lower mortality percent range for polyDADMAC and TCMTB and in the upper mortality percent range for niclosamide ethanolamine salt, in which cases TUx,i values were equal to or slightly above one. Generally speaking, the action of the four biocides can thus be said to have been synergised as they were mixed in the ratio of their median lethal concentrations. In the case of LC10 formulation (Fig. 4b), the actions of potassium chloride, polyDADMAC and TCMTB were synergised over the entire response level range as they were incorporated in the combination. However, from the comparison between Figs. 4a and b, major differences in the performance of niclosamide ethanolamine salt are evident. In LC10 formulation, the action of this molluscicide was synergised in mixtures producing low to intermediate mortality percents, but it was observed to undergo significant antagonistic effects at response levels above 60%, with an individual number of toxic units of almost 2 for a percent response of 90%. It is worth nothing that the differences between the two formulations in terms of the variation of the individual chemicals' toxicity (that is, in the relative contributions of TUx,i values to the total TUx,mix) reflect the difference between the chemicals' relative proportions in the formulations (Table 1) because the blends' dose–response curves (LCx,i) were similar (Fig. 2) and, by p ⋅LC definition, TUx,i is given by the quotient i LC x;ix;mix . Central composite design toxicity tests The bioassays involving the testing of a series of mixtures of varying composition following a central composite experimental design did not provide meaningful results. The coefficient of determination characterising the second-order response surface model fitted to the results was 0.589 (p = 0.365). None of the model coefficients were found to be statistically significant (p values between 0.088 and 0.889), and therefore it was not possible to definitely infer the relative contribution of each biocide and that of their interactions to the mixture toxicity. Discussion Potential of the quaternary mixture for zebra mussel control

Fig. 1. Dose–response data for the single molluscicides: (a) exposure to potassium chloride for 48 h; (b) exposure to polyDADMAC for 36 h; (c) exposure to niclosamide ethanolamine salt for 36 h; (d) exposure to TCMTB for 36 h. The points refer to the experimental mortality data (mean ± SE); the lines represent models obtained by Probit analysis (p ≤ 0.02).

df = 8; p = 0.002), but were always significantly higher than one (p b 0.05). Such values were not significantly affected by the formulation ratio (two-factor ANOVA; F = 0.188; df = 1; p = 0.676). In Fig. 4, TUx,mix values producing lethal responses in the range 10 to 90% are presented as the sum of the number of toxic units of the mixture ingredients (TUx,i) as established by Eq. (1), allowing for the potential of the formulations to be further assessed by referring to the

The results obtained in the single substance bioassays (Fig. 1 and Table 3) are consistent with the toxicity data reported in other studies (see, for example, Fisher et al., 1991; McMahon et al., 1993; Waller et al., 1993; Durand-Hoffman, 1995; and more recently Costa et al., 2011). Mixtures of the four molluscicides do not seem to have been tested before, and therefore a comparison of the results obtained in the integral mixture toxicity tests (Fig. 2) with literature data is not possible. As far as the overall mixture performance is concerned, both LC50 formulation and LC10 formulation were found to be less than additive over the entire mortality range, with TUx,mix values consistent with what has generally been reported in ecotoxicological studies (Fig. 3). Several authors (ECETOC, 2001; Warne, 2002) have pointed out that there are not many examples in the literature of blends whose toxicity differs from concentration addition by a factor greater than three. The toxic nature of the quaternary combination as a whole did not depend on the ratio at which the components were mixed (Fig. 3). Note that this was the case even though the compositions of LC50 formulation and LC10 formulation were substantially different (Table 1). As the chemicals were present at the same proportion of their individual toxicities in the two blends, it is likely that this behaviour is representative of that of mixtures in which no single ingredient accounts for an overwhelming proportion of mixture toxicity. In general, the joint action of chemicals may depend on the mixture's quantitative

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Table 3 Selected lethal concentrations of potassium chloride, polyDADMAC, niclosamide ethanolamine salt and TCMTB for the duration of the individual treatments (48 h in the case of potassium chloride and 36 h in the case of the remaining biocides). The estimated concentrations and respective 95% confidence interval limits were obtained by Probit analysis. Biocide

Potassium chloride PolyDADMAC Niclosamide ethanolamine salt TCMTB

Median lethal concentration after the end of the treatment (mg/l)

Lethal concentration for percentile 10 after the end of the treatment (mg/l)

Estimate

95% confidence interval

Estimate

95% confidence interval

247 61.4 0.177 243

133–464 40.0–92.6 0.153–0.199 89.5–727

87.0 5.06 0.0962 24.3

30.6–217 1.68–9.86 0.0711–0.116 4.72–95.9

composition (Warne, 2002). However, several cases of combinations whose overall toxic character was not affected by the relative proportion of their components have been reported in the literature (e.g. Faust et al., 2001).

In addition to characterising the biocides' overall joint action, the results of the integral mixture bioassays were also analysed in terms of the variation of the chemicals' individual lethality as they were combined. Such variation was observed to depend on the ratio at which the chemicals were blended as well as the magnitude of the response under consideration (Fig. 4). Remarkably, even though the sensitivity of the mussels to niclosamide ethanolamine salt seemed to diminish as it was incorporated in LC10 formulation, the two tested blends as a whole produced similar dose–response curves (Fig. 2) and they were characterised by similar TUx,mix values (Fig. 3). The detailed reasons for this cannot be drawn from this study. However, a possible

Fig. 2. Dose–response data for LC50 formulation and LC10 formulation. The points refer to the experimental mortality data (mean ± SE); the lines represent dose–response models obtained by Probit analysis (p ≤ 0.001).

Fig. 3. Joint toxicity of potassium chloride, polyDADMAC, niclosamide ethanolamine salt and TCMTB, combined at different ratios in LC50 formulation and LC10 formulation, at varying mortality levels. The solid lines represent the estimated TUx,mix and the dashed lines represent the respective 95% confidence interval limits obtained by Probit analysis.

Fig. 4. Variation of the individual toxicity of potassium chloride, polyDADMAC, niclosamide ethanolamine salt and TCMTB as they were combined in (a) LC50 formulation or (b) LC10 formulation. This variation is expressed in terms of TUx,i in the mixtures producing responses in the range 10 to 90%. The width of the shaded regions represents the magnitude of the TUx,i values; the total graph area corresponds to the sum of the TUx,i values, which is, by definition, TUx,mix shown in Fig. 3.

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interpretation for this result is as follows. It is accepted that the mode of action of toxicants upon an organism may change with varying exposure concentrations (Könemann and Pieters, 1996; Groten et al., 2001; Barata et al., 2006). As the ratio, and consequently the test range, of the toxin concentrations varied from LC50 formulation to LC10 formulation, the toxicity mechanisms of the biocides and/or the interactions between them may also have varied. It is conceivable that these variations occurred in such a way that the action of niclosamide ethanolamine salt was hampered in the LC10 formulation, but the actions of the other biocides (in particular polyDADMAC and TCMTB) were more favoured in this formulation than in LC50 formulation (Fig. 4). As a result, the overall combination toxicity was similar in both cases. In other words, as the compounds were mixed in the ratio of their lethal concentrations for percentile 10, their biological actions and interactions varied in such a way that a unitary concentration of niclosamide ethanolamine salt became less lethal than in the LC50 formulation, but a unitary concentration of the other components became more toxic so that the two formulations as a whole produced similar effects. The results discussed above seriously compromise the potential of quaternary mixtures of potassium chloride, polyDADMAC, niclosamide ethanolamine salt and TCMTB for practical zebra mussel control, with two points deserving to be highlighted in this context. The first is the fact that, by being less than additive, the quaternary mixture does not provide an effective reduction of the total toxic load required for a certain level of pest mitigation. Formulating less than additive mixtures, in practice, corresponds to totally or partially replacing one of the ingredients by a less toxic fraction of another. The second point that is worth discussing with regard to the potential of the mixture for pest control concerns the fact that synergistic effects were observed under certain circumstances, meaning that a given mortality percent was elicited by a biocide dosage lower than that required when the remaining chemicals were not present. This effect was particularly noticeable for polyDADMAC and TCMTB at high response levels, which are those of major interest for practical control (Fig. 4). Due to such synergistic action, the quaternary mixture could possibly be suggested as beneficial for use in situations where the reduction of the application requirements of one of these toxins is intended. Note, however, that at the target response range the synergism of polyDADMAC and TCMTB actions was achieved at the expense of slight (Formulation LC50; Fig. 4a) to significant (Formulation LC10; Fig. 4b) antagonism of niclosamide ethanolamine salt activity. Moreover, it is likely that similar or even greater increases of polyDADMAC and TCMTB toxicities may be achieved through simpler mixtures. Within this context, the reduced potential of the quaternary mixture can be illustrated by comparing the results obtained in this study with those presented by Costa et al. (2011), who showed that potassium chloride alone significantly synergises polyDADMAC action. As shown in Fig. 5, the susceptibility of adult zebra mussels to combined potassium chloride and polyDADMAC together decreased in the presence of niclosamide ethanolamine salt and TCMTB. Pest control through the quaternary mixture instead of the binary blend would then be disadvantageous, implying not only the application of higher potassium chloride and polyDADMAC concentrations, but also the dosing of additional biocides into the industrial plants.

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Fig. 5. Comparison of the average toxicity for binary (Costa et al., 2011) and quaternary (this study) mixtures containing potassium chloride and polyDADMAC.

to the toxicity data obtained (Box and Draper, 1987; Montgomery and Runger, 1999). Ideally, this analysis would have resulted in an empirical model enabling the prediction of the mixture toxicity from the concentration of its components, which is impossible to obtain mechanistically (as illustrated by Fig. 6). The magnitude of the empirical model coefficients associated with the linear and quadratic toxin concentrations would have provided information on the contribution of the biocides to the combined effect whilst the magnitude of the coefficient of the product terms would have captured the role played by the interactions between pairs of chemicals. Unfortunately, the experimental results did not allow a meaningful mixture toxicity model to be obtained due to the high background variation of the response to the treatments. This type of variation is not uncommon, and other authors have experienced similar difficulties in ecotoxicological studies. For example, for the same reasons, Lock and Janssen (2002) did not succeed in developing

Individual contributions to the mixture performance and modelling of the chemicals' joint action The results of the integral mixture toxicity study did not provide information on the contribution of each biocide to the observed mixture performance nor on the possibility of optimising the combination effects. In an attempt to obtain this type of information, several mixtures were tested following a central composite experimental design, and a second-order response surface model was fitted

Fig. 6. Assessment of the ability of concentration addition and response addition concepts to predict the toxicity of LC50 formulation. The points refer to the experimental dose–response data presented in Fig. 2 and the curves represent the estimated dose–response relationships. For further details on the mechanistic models of joint toxicity see, for example, Könemann and Pieters (1996), Faust et al. (2001), Backhaus et al. (2003) and de Zwart and Posthuma (2005).

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an empirical model for the combined toxicity of zinc, cadmium, copper and lead to the potworm Enchytraeus albidus. General approach, multivariate experiments and empirical modelling in multicomponent molluscicide mixture design Although the empirical modelling of the blend toxicity has not been successful in this study, the value of using multivariate statistical techniques in the design of multicomponent mixtures for zebra mussel control should not be neglected as discussed below. The first main challenge a pest manager faces when designing a combination of biocides is to define its qualitative composition. Preliminary selection of candidate ingredients will usually be based on both the known chemicals' toxic activity and specific system requirements. The number of compounds chosen at this initial stage may still be prohibitively large to explore in detail their joint action. Furthermore, there are two issues that have to be considered when deciding on the complexity of the molluscicide formulation. First, ecotoxicological studies on mixture toxicity suggest that there is a tendency for blends to be additive as the number of components increases (Warne and Hawker, 1995; Warne, 2002) whilst more than additive combinations are more beneficial from the control perspective. Second, potential operational and effluent purification problems related to the dosage of a large number of substances into the system have to be outweighed by the enhanced performance of multiple biocides. For these reasons, less complex biocide mixtures are usually preferable in practice. A systematic screening of the preliminary set of molluscicide candidates is thus useful in order to identify those that contribute the most to mixture performance as well as undesirable antagonistic interactions. In this context, two-level full factorial or fractional factorial experimental designs (Montgomery and Runger, 1999) may prove valuable. Once a promising set of chemicals is identified, the nature of their joint action can then be described by following the systematic approach proposed in this paper in order to assess the potential of the formulation. The qualitative composition of the mixture may be iteratively re-adjusted at any stage of the design process as the understanding of the behaviour of the biocides in the presence of each other increases. Another important challenge in designing molluscicide combinations for zebra mussel control is to be able to mathematically relate the mixture performance to the concentration of the individual ingredients. A model of this type may be integrated in an optimisation framework, so that the final composition of the formulation is set to provide optimal performance under system-specific economic, regulatory and environmental constraints. Typically, a mechanistic approach will not prove adequate to derive a model for mixture toxicity, because the concepts of concentration addition and response addition are often of limited use in practice (Könemann and Pieters, 1996; van der Geest et al., 2000; de Zwart and Posthuma, 2005; Barata et al., 2006). Therefore, the use of multivariate statistical tools for empirical modelling, such as response surface regression analysis (Box and Draper, 1987; Montgomery and Runger, 1999; Lock and Janssen, 2002), will be the preferred approach to obtain blend toxicity models. Practical implications of the study Even though mixtures of potassium chloride, polyDADMAC, niclosamide ethanolamine salt and TCMTB were observed to synergise the action of some of the ingredients, the quaternary combination is not recommended for zebra mussel control. Although this major result does not directly lead to an innovative approach to pest mitigation, the present study highlighted some aspects of practical relevance, which are useful in the general context of designing multicomponent molluscicide mixtures.

One of such aspects arises from the overview of the ecotoxicological literature on mixture toxicity. As stated by the funnel hypothesis, there is a propensity for multiple chemicals to act additively as the number of ingredients in the mixture increases (Warne and Hawker, 1995; Warne, 2002). This provides key guidance in the process of designing a new molluscicide blend. As more than additive combinations are those that should be achieved, there is no advantage in formulating excessively complex mixtures. This study also highlights the fact that biocide cocktails do not necessarily increase the control effectiveness, even though this may be intuitively expected. The mixture may produce mortalities higher than those elicited by its individual ingredients but still exert a less than additive action, which is not particularly beneficial from the control point of view. The overall mixture toxicity may also result from the synergism of the action of some components at the expense of the antagonism of others. Under these circumstances, the effects elicited by the mixture could be equally achieved by applying only one of the antagonised chemicals at a dosage lower than its concentration in the blend. Finally, as a holistic practical contribution, this paper has also proposed a general approach that may be employed to design multicomponent mixtures for zebra mussel control. Acknowledgments Financial support from the Portuguese Foundation for Science and Technology (PhD scholarship SFRH/BD/18731/2004 and research project POCI/EQU/59305/2004) is gratefully acknowledged. References Ahmad, M., 2004. Potentiation/antagonism of deltamethrin and cypermethrins with organophosphate insecticides in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae). Pestic. Biochem. Physiol. 80, 31–42. Aldridge, D.C., Elliott, P., Moggridge, G.D., 2006. Microencapsulated BioBullets for the control of biofouling zebra mussels. Environ. Sci. Technol. 40, 975–979. Andrews, P., Thyssen, J., Lorke, D., 1982. The biology and toxicology of molluscicides. Bayluscide. Pharmacol. Ther. 19, 245–295. Backhaus, T., Altenburger, R., Boedeker, W., Faust, M., Scholze, M., Grimme, L.H., 2000. Predictability of the toxicity of a multiple mixture of dissimilarly acting chemicals to Vibrio fischeri. Environ. Toxicol. Chem. 19, 2348–2356. Backhaus, T., Altenburger, R., Arrhenius, A., Blanck, H., Faust, M., Finizio, A., Gramatica, P., Grote, M., Junghans, M., Meyer, W., Pavan, M., Porsbring, T., Scholze, M., Todeschini, R., Vighi, M., Walter, H., Grimme, L.H., 2003. The BEAM-project: prediction and assessment of mixture toxicities in the aquatic environment. Cont. Shelf Res. 23, 1757–1769. Barata, C., Baird, D.J., Nogueira, A.J., Soares, A.M., Riva, M.C., 2006. Toxicity of binary mixtures of metals and pyrethroid insecticides to Daphnia magna Straus. Implications for multi-substance risks assessment. Aquat. Toxicol. 78, 1–14. Box, G.E.P., Draper, N.R., 1987. Empirical Model-building and Response Surfaces. Wiley, Chichester. Claudi, R., Mackie, G.L., 1994. Practical Manual for Zebra Mussel Monitoring and Control. Lewis Publishers, Boca Raton. Costa, R., Aldridge, D.A., Moggridge, G.D., 2008. Seasonal variation of zebra mussel susceptibility to molluscicidal agents. J. Appl. Ecol. 45, 1712–1721. Costa, R., Elliott, P., Aldridge, D.C., Moggridge, G.D., 2011. Enhanced mortality of the biofouling zebra mussel, Dreissena polymorpha, through the application of combined control agents. J. Great Lakes Res. 37, 272–278. de Zwart, D., Posthuma, L., 2005. Complex mixture toxicity for single and multiple species: proposed methodologies. Environ. Toxicol. Chem. 24, 2665–2676. Durand-Hoffman, M.E., 1995. Analysis of physiological and toxicological effects of potassium on Dreissena polymorpha and toxicological effects on fish. MSc thesis. Ohio State University, Columbus, US. ECETOC, 2001. Aquatic toxicity of mixtures. Technical Report 80. European Centre for Ecotoxicology and Toxicology of Chemicals, Brussels. Eide, I., 1996. Strategies for toxicological evaluation of mixtures. Food Chem. Toxicol. 34, 1147–1149. Elliott, P., Aldridge, D.C., Moggridge, G.D., Chipps, M., 2005. The increasing effects of zebra mussels on water installations in England. Water Environ. J. 19, 367–375. Elzinga, W.J., Butzlaff, T.S., 1994. Carbon dioxide as a narcotizing pre-treatment for chemical control of Dreissena polymorpha. Paper Presented at Fourth International Zebra Mussel Conference. ICAIS, Madison. Faust, M., Altenburger, R., Backhaus, T., Blanck, H., Boedeker, W., Gramatica, P., Hamer, V., Scholze, M., Vighi, M., Grimme, L.H., 2001. Predicting the joint algal toxicity of multicomponent s-triazine mixtures at low-effect concentrations of individual toxicants. Aquat. Toxicol. 56, 13–32.

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