**3. Results**

#### **3.1. Responses to treatment with chlorpyrifos and dimethoate**

Principal Components Analysis (PCA) of the 38 genera and 5 orders of benthic macroinver‐ tebrates identified in this experiment were highly responsive to increasing TU treatment and responded differently to treatment with either chlorpyrifos or dimethoate (Figure 4). Factor 1, (Eigenvalue 7.08, 44.3% of variance) was composed of the combined loadings of treatment in toxic units (TU, Pearson's *r* = 0.34) as well as the action of chlorpyrifos (Pearson's *r* = 0.58) or dimethoate (Pearson's *r* = -0.15). Increased insecticide treatment in Toxic Units (TU) re‐ duced the breadth of taxa present in the community assemblage, as indicated by the de‐ creased variation in the distribution of taxa and guilds from left to right along the horizontal axis (Factor 1 in Figure 4). Interestingly, community responses to treatment with either chlorpyrifos or dimethoate were in opposing directions, although both insecticides were im‐ portant contributors to the distribution of taxa, guilds and treatments in Factor 2 (Eigenval‐ ue 2.36, 14.7%; TU, Pearson's *r* = 0.01; chlorpyrifos, Pearson's *r* = -0.31; dimethoate, Pearson's *r* = 0.29). In particular, chlorpyrifos was an important contributor to the removal of taxa with streams treated with 0.8 TU of chlorpyrifos (C0.8TU) occurring in the PCA quadrant with the fewest taxa (bottom right, Figure 4). By contrast, responses to treatment with dimethoate occurred in the opposite quadrant suggesting firstly, that different members of the benthic macroinvertebrate assemblage were responding to chlorpyrifos versus dimethoate, and that treatment with dimethoate did not decrease density and diversity of taxa as forcefully as treatment with chlorpyrifos (top left, Figure 4). Interestingly, medium dose mixture treat‐ ments (M0.4TU) are located in the same quadrant as the equivalent dimethoate treatments (e.g., D0.4TU and D0.8TU) whereas high dose mixtures (M0.8TU) were more closely associ‐ ated with predictions of additive toxicity in toxic units (Factor 1).

concentrations of insecticides) as well as the density of in-stream macroinvertebrates [19]. A correlation matrix was used to prevent the different variances in the variables to influence the analysis. Responses in different taxa and guilds were also examined using factorial AN‐

factorial ANOVA approaches examined response variables with respect to explicit treat‐ ment categories: a gradient of toxic units (TU, throughout); different insecticide treatments (I) and the interaction between the dose and the insecticide treatments (TU x I). Post-hoc testing, where applicable, was conducted using 1-tailed Dunnett's tests [20] and compared specific treatments to control levels (ANOVA approach, marked 'a' in corresponding fig‐ ures). Where necessary (e.g., total and scraper abundance), data were transformed to satisfy assumptions (ln transformation, [21]). Whether the treatments initiated predictable reduc‐ tions in abundance (of taxa, groups or guilds) was examined by comparing observed differ‐

determined by calculating the predicted reduction compared to control values for each in‐ vertebrate metric, in abundance from the toxic unit treatment range. Predicted values with respect to control appear throughout and significant deviations from predicted values by the χ<sup>2</sup> approach are marked 'c' in the corresponding figures. Preliminary comparisons of dif‐ ferences between the low binary (0.1 TU x 2) and low ternary (0.1 TU x 3) mixtures (1-way ANOVA) are also made for the six response variables of interest with respect to control, pre‐ dicted, binary and ternary mixture treatment levels. To simplify, although differences in

Principal Components Analysis (PCA) of the 38 genera and 5 orders of benthic macroinver‐ tebrates identified in this experiment were highly responsive to increasing TU treatment and responded differently to treatment with either chlorpyrifos or dimethoate (Figure 4). Factor 1, (Eigenvalue 7.08, 44.3% of variance) was composed of the combined loadings of treatment in toxic units (TU, Pearson's *r* = 0.34) as well as the action of chlorpyrifos (Pearson's *r* = 0.58) or dimethoate (Pearson's *r* = -0.15). Increased insecticide treatment in Toxic Units (TU) re‐ duced the breadth of taxa present in the community assemblage, as indicated by the de‐ creased variation in the distribution of taxa and guilds from left to right along the horizontal axis (Factor 1 in Figure 4). Interestingly, community responses to treatment with either chlorpyrifos or dimethoate were in opposing directions, although both insecticides were im‐ portant contributors to the distribution of taxa, guilds and treatments in Factor 2 (Eigenval‐ ue 2.36, 14.7%; TU, Pearson's *r* = 0.01; chlorpyrifos, Pearson's *r* = -0.31; dimethoate, Pearson's *r* = 0.29). In particular, chlorpyrifos was an important contributor to the removal of taxa with streams treated with 0.8 TU of chlorpyrifos (C0.8TU) occurring in the PCA quadrant with the fewest taxa (bottom right, Figure 4). By contrast, responses to treatment with dimethoate occurred in the opposite quadrant suggesting firstly, that different members of the benthic

were tested for significance, the responses are shown as the percent reduc‐

) approaches. In this study,

) tests. Expected values were

OVA (for chlorpyrifos and dimethoate only) and chi-square (χ<sup>2</sup>

90 Insecticides - Development of Safer and More Effective Technologies

ences to those expected (or predicted) using chi-square (χ<sup>2</sup>

tion in response between the ternary and the binary mixtures at 0.1 TU.

**3.1. Responses to treatment with chlorpyrifos and dimethoate**

density per cm2

**3. Results**

**Figure 4.** Principal Components Analysis (PCA) of differences in responses of 38 genera and 5 orders of benthic mac‐ roinvertebrates (each indicated, •) associated with chlorpyrifos or dimethoate insecticide treatment in Toxic Units (as vectors, above). Each treatment level is indicated (e.g., C0.2 TU, Chlorpyrifos at 0.2 TU). Factor 1 explained 44.3 % of the variance in the assemblages and was primarily driven by increased insecticide treatment in Toxic Units and secon‐ darily by chlorpyrifos treatment. Dimethoate treatment was associated with different assemblages predominantly contributing to pattern in Factor 2 which explained an additional 14.7 % of the variance. Additional notes: guilds are indicated by codes cf = collector-filterers; cg = collector-gatherers; sc = scrapers; sh = shredders; pr = predators; total abundance per cm2 = N; total richness per cm2 = s; E.P.T. = sum density of Ephemeroptera, Plecoptera and Trichoptera orders. Remaining labels indicate genera of aquatic insect taxa (e.g., *Chironomus* spp.).

Significant change in measures of average total density per cm2 and average taxa richness per cm2 (Figure 5) were only found at the highest dose of chlorpyrifos tested (0.8 TU, abun‐ dance or richness, *P* < 0.01). The highly significant interactions (total density, TU x I, *F5, 69 =* 68.23, *P <* 0.01; or richness, TU x I, *F5, 69 =* 709.03, *P <* 0.01) were the result of total density and richness being decreased as predicted under exposure to chlorpyrifos, while dimethoate had no such effect. Throughout this study, dimethoate was non-toxic with respect to total densi‐ ty and richness and no negative effects of insecticides were detected irrespective of dose. Additionally, mixture treatments were not different than control levels for either total densi‐ ty or richness (e.g., total density in M0.8TU, *P =* 0.97; richness in M0.8TU, *P =* 0.75). Stream communities were significantly more dense than predicted in high dose treatments contain‐ ing dimethoate including the high mixture (M0.8TU, *χ<sup>2</sup> <sup>7</sup> =* 20.24, *P* < 0.01) and the high di‐ methoate treatment (D0.8TU, *χ<sup>2</sup> <sup>7</sup> =* 16.90, *P* < 0.01). In contrast, taxa richness was not found to be significantly different than predicted.

Scraper density was not different than the control, although predators were highly respon‐ sive to all high dose insecticide treatments (*P <* 0.01, Figure 6). Once again, significant inter‐ actions were found for both guilds (scrapers, TU x I, *F5, 69 =* 12.46, *P <* 0.01; predators, TU x I, *F5, 69 =* 26.35, *P <* 0.01). However, the extent of significant interactions in scraper genera ap‐ peared to be largely due to the high variation in the density of the guild in the low dose, chlorpyrifos treatment (0.2 TU). Doses of 0.2 to 0.4 TU of chlorpyrifos and 0.2 TU of dime‐ thoate all contained more scrapers than predicted (e.g., 74 % greater than predicted scraper density in chlorpyrifos 0.2 TU, *χ<sup>2</sup> <sup>7</sup> =* 50.03, *P* < 0.01). In contrast, responses in predators were unique in that they responded to high insecticide doses (0.8 TU) by significantly decreasing abundance in these treatments, irrespective of the insecticide applied (e.g., 0.8TU mixture, 46 % less than predicted, *χ<sup>2</sup> <sup>7</sup> =* 28.38, *P* < 0.01). Finally, the bell-shaped abundance pattern in predators with increased dimethoate treatment, compared with the linear decrease in abun‐ dance of the chlorpyrifos treatment, suggests that responses in predators were more com‐ plex than in other groups, potentially as a result of indirect effects due to reduced prey

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**Figure 6.** Density of E.P.T., Chironomus spp., scrapers and predators per cm2 (± 1 SE, n = 8) compared to treatment with the insecticides chlorpyrifos (black bars), dimethoate (white bars) or a 1:1 mixture of both insecticides (patterned bars). Letters indicate: 'a' significant differences compared to control (ANOVA approach), and 'c' differences in specific

density.

treatments (χ2 approach).

**Figure 5.** Total abundance and richness per cm2 (± 1 SE, n = 8) of aquatic macroinvertebrates compared to treatment with the insecticides chlorpyrifos (black bars), dimethoate (white bars) or a 1:1 mixture of both insecticides (patterned bars). Letters indicate: 'a' significant differences compared to control (ANOVA approach), and 'c' differences in specific treatments (χ2 approach).

Responses in the average density of E.P.T. taxa and *Chironomus* spp. per cm2 (Figure 6) were only found to significantly differ from control values in the highest chlorpyrifos treatment level (0.8 TU, E.P.T. or *Chironomus*, *P <* 0.01). Highly significant interactions were evident (E.P.T., TU x I, *F5, 69 =* 53.91, *P <* 0.01; or *Chironomus*, TU x I, *F5, 69 =* 50.02, *P <* 0.01) because density of E.P.T. and *Chironomus* decreased due to chlorpyrifos but not due to dimethoate. However, *Chironomus* midges were highly negatively affected by 0.8 TU of chlorpyrifos and the mean density of larvae in this treatment level was reduced 96% compared to controls (predicted decrease at 0.8 TU = 40%; C0.8TU, *χ<sup>2</sup> <sup>7</sup> =* 31.45, *P* < 0.01). E.P.T. taxa were highly sensitive to high dose treatment with chlorpyrifos (C0.8TU, *χ<sup>2</sup> <sup>7</sup> =* 12.75, *P* < 0.01), however, treatments containing dimethoate (e.g., dimethoate and mixture) were much less toxic than predicted (e.g., mean E.P.T. density in 0.8TU mixture, 37 % greater than predicted).

Scraper density was not different than the control, although predators were highly respon‐ sive to all high dose insecticide treatments (*P <* 0.01, Figure 6). Once again, significant inter‐ actions were found for both guilds (scrapers, TU x I, *F5, 69 =* 12.46, *P <* 0.01; predators, TU x I, *F5, 69 =* 26.35, *P <* 0.01). However, the extent of significant interactions in scraper genera ap‐ peared to be largely due to the high variation in the density of the guild in the low dose, chlorpyrifos treatment (0.2 TU). Doses of 0.2 to 0.4 TU of chlorpyrifos and 0.2 TU of dime‐ thoate all contained more scrapers than predicted (e.g., 74 % greater than predicted scraper density in chlorpyrifos 0.2 TU, *χ<sup>2</sup> <sup>7</sup> =* 50.03, *P* < 0.01). In contrast, responses in predators were unique in that they responded to high insecticide doses (0.8 TU) by significantly decreasing abundance in these treatments, irrespective of the insecticide applied (e.g., 0.8TU mixture, 46 % less than predicted, *χ<sup>2</sup> <sup>7</sup> =* 28.38, *P* < 0.01). Finally, the bell-shaped abundance pattern in predators with increased dimethoate treatment, compared with the linear decrease in abun‐ dance of the chlorpyrifos treatment, suggests that responses in predators were more com‐ plex than in other groups, potentially as a result of indirect effects due to reduced prey density.

Additionally, mixture treatments were not different than control levels for either total densi‐ ty or richness (e.g., total density in M0.8TU, *P =* 0.97; richness in M0.8TU, *P =* 0.75). Stream communities were significantly more dense than predicted in high dose treatments contain‐ ing dimethoate including the high mixture (M0.8TU, *χ<sup>2</sup> <sup>7</sup> =* 20.24, *P* < 0.01) and the high di‐ methoate treatment (D0.8TU, *χ<sup>2</sup> <sup>7</sup> =* 16.90, *P* < 0.01). In contrast, taxa richness was not found

**Figure 5.** Total abundance and richness per cm2 (± 1 SE, n = 8) of aquatic macroinvertebrates compared to treatment with the insecticides chlorpyrifos (black bars), dimethoate (white bars) or a 1:1 mixture of both insecticides (patterned bars). Letters indicate: 'a' significant differences compared to control (ANOVA approach), and 'c' differences in specific

only found to significantly differ from control values in the highest chlorpyrifos treatment level (0.8 TU, E.P.T. or *Chironomus*, *P <* 0.01). Highly significant interactions were evident (E.P.T., TU x I, *F5, 69 =* 53.91, *P <* 0.01; or *Chironomus*, TU x I, *F5, 69 =* 50.02, *P <* 0.01) because density of E.P.T. and *Chironomus* decreased due to chlorpyrifos but not due to dimethoate. However, *Chironomus* midges were highly negatively affected by 0.8 TU of chlorpyrifos and the mean density of larvae in this treatment level was reduced 96% compared to controls (predicted decrease at 0.8 TU = 40%; C0.8TU, *χ<sup>2</sup> <sup>7</sup> =* 31.45, *P* < 0.01). E.P.T. taxa were highly sensitive to high dose treatment with chlorpyrifos (C0.8TU, *χ<sup>2</sup> <sup>7</sup> =* 12.75, *P* < 0.01), however, treatments containing dimethoate (e.g., dimethoate and mixture) were much less toxic than

(Figure 6) were

Responses in the average density of E.P.T. taxa and *Chironomus* spp. per cm2

predicted (e.g., mean E.P.T. density in 0.8TU mixture, 37 % greater than predicted).

to be significantly different than predicted.

92 Insecticides - Development of Safer and More Effective Technologies

treatments (χ2 approach).

**Figure 6.** Density of E.P.T., Chironomus spp., scrapers and predators per cm2 (± 1 SE, n = 8) compared to treatment with the insecticides chlorpyrifos (black bars), dimethoate (white bars) or a 1:1 mixture of both insecticides (patterned bars). Letters indicate: 'a' significant differences compared to control (ANOVA approach), and 'c' differences in specific treatments (χ2 approach).

#### **3.2. Preliminary findings comparing binary and ternary mixtures**

Statistical comparisons of the differences in density between binary (0.1 TU x 2) and ternary (0.1 TU x 3) mixtures of insecticides determined that the average total density (*P = 0.02*), taxa richness (*P < 0.01*) and *Chironomus* spp. (*P < 0.01*) were all significantly reduced due to the addition of imidacloprid to the mixture (Figure 7). In contrast, the average density of E.P.T. genera, scrapers and predators were not found to be significantly reduced in the presence of imidacloprid (*P > 0.06*, all cases). On average, the addition of a third insecticide resulted in a 62.9 ± 13.0 % reduction in average density. Density was more greatly reduced in some groups than others with scrapers the most affected (-111.6 ± 16.9 %) and taxa richness the least affected (-18.2 ± 16.5 %).

oxygen analogue (oxon) via metabolic bioactivation, creating an excretable endproduct which is also potentially toxic [9]. It is the oxon that binds acetylcholinesterase (AChE) and prevents the capture and removal of acetylcholine in the synapses, creating a positive feed‐ back loop whereby uncontrolled neural signalling is initiated. Therefore, increased or de‐ creased toxicity, even from the standpoint of a single mode of action (AChE), is due to the interaction of at least five factors: firstly, in/efficient creation of the oxygen analogue (oxon), i.e., differences in basal metabolism; secondly, insufficient binding of the target esterase(s) and/or binding to alternative targets; thirdly, insufficient accumulation of acetylcholine in the synaptic gap, due to inherent neurochemical differences or deficiencies, e.g., Myasthenia gravis; fourthly, other forms of tolerance and/or resistance, e.g., species, strain or regional differences (e.g., as reported in [22]), and finally, excretion and/or uptake efficiency of the parent toxicant or its metabolites. Furthermore, organophosphates also bind other receptors (e.g., muscarinic and nicotinic receptors), which in themselves can up or down regulate the

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Despite the equivalent toxic unit doses employed in this study, treatment with dimethoate was associated with increased abundance of different taxa and guilds with the exception of predators, which were found to be substantially negatively impacted by all high dose treat‐ ments. In mixture treatments, the density of taxa often fell between that of either of the two insecticides individually, or, resembled the relatively non-toxic dimethoate at 0.4 and 0.8 TU. The highly significant declines in abundance of different taxa and guilds due to chlor‐ pyrifos treatment, and the lack of similar findings due to dimethoate treatment are troubling because this study determined the appropriate doses from standard bioassays of the same genera from public databases of the published literature. For instance, according to a Nor‐ wegian study by Baekken and Aanes [5], the 96-hr LC50 for *Baetis rhodani* exposed to dime‐ thoate was ~ 7 µg/L. In this study *Baetis* not only survived but emerged as adults (37 females and 26 males, not shown) in the 0.8 TU treatment where the dimethoate concentration was in the range of 19.93 – 22.96 µg/L. Disparities such as these invite speculation. If regional dif‐ ferences in sensitivity are as pronounced as the above finding suggests, then modeling may be restricted to more local scales. Alternatively, regional variation in data quality also invites

This study generally found that the mixture pattern at high doses had intermediate toxicity. Specifically, invertebrate responses to the binary mixtures were between that of dimethoate or chlorpyrifos individually. LeBlanc *et al.* [14] also found mixtures of chlorpyrifos and di‐ methoate to exhibit dose-level dependency in concurrent laboratory studies using chlorpyri‐ fos and dimethoate in both binary mixtures (i.e., low dose antagonism to high dose synergy). Although high dose exposures are likely less common than sublethal effects (as described in [24]), high dose synergy is a concern because isolated high-dose events (e.g., a rain event) could significantly alter the composition of aquatic communities. Additionally, in more complex mixtures where multiple modes of action may be the norm, the concentra‐ tion that initiates a synergistic effect may be lower than implied from bioassay results using

effectiveness of the insecticide dose [23].

single-species and single compounds.

speculation.

**Figure 7.** Comparison of % reduction in metrics due to treatment with the ternary mixture of 0.1 TU versus the binary mixture with the same doses. Each 0.1 TU dose should reduce the density of sensitive taxa by 5% because 1 TU = LC50. Therefore, reductions greater than 5% in the density of aquatic taxa is of biological interest even if differences in the density of organisms were not found to be statistically significant.

#### **4. Discussion**

#### **4.1. Responses to chlorpyrifos and dimethoate**

All of the metrics of benthic invertebrate responses measured also had significant interaction terms (TU x I, *P <* 0.1) suggesting that not all taxa, groups or guilds were equally sensitive to insecticide treatment. Differential toxicity within the organophosphorus insecticides has been reported previously and is predominantly due to the complexity of the biochemical pathway to reach what is considered the primary target, acetylcholinesterase [8,9,10]. Specif‐ ically, the toxic potency of organophosphorus insecticides depends on the creation of an oxygen analogue (oxon) via metabolic bioactivation, creating an excretable endproduct which is also potentially toxic [9]. It is the oxon that binds acetylcholinesterase (AChE) and prevents the capture and removal of acetylcholine in the synapses, creating a positive feed‐ back loop whereby uncontrolled neural signalling is initiated. Therefore, increased or de‐ creased toxicity, even from the standpoint of a single mode of action (AChE), is due to the interaction of at least five factors: firstly, in/efficient creation of the oxygen analogue (oxon), i.e., differences in basal metabolism; secondly, insufficient binding of the target esterase(s) and/or binding to alternative targets; thirdly, insufficient accumulation of acetylcholine in the synaptic gap, due to inherent neurochemical differences or deficiencies, e.g., Myasthenia gravis; fourthly, other forms of tolerance and/or resistance, e.g., species, strain or regional differences (e.g., as reported in [22]), and finally, excretion and/or uptake efficiency of the parent toxicant or its metabolites. Furthermore, organophosphates also bind other receptors (e.g., muscarinic and nicotinic receptors), which in themselves can up or down regulate the effectiveness of the insecticide dose [23].

**3.2. Preliminary findings comparing binary and ternary mixtures**

94 Insecticides - Development of Safer and More Effective Technologies

least affected (-18.2 ± 16.5 %).

Statistical comparisons of the differences in density between binary (0.1 TU x 2) and ternary (0.1 TU x 3) mixtures of insecticides determined that the average total density (*P = 0.02*), taxa richness (*P < 0.01*) and *Chironomus* spp. (*P < 0.01*) were all significantly reduced due to the addition of imidacloprid to the mixture (Figure 7). In contrast, the average density of E.P.T. genera, scrapers and predators were not found to be significantly reduced in the presence of imidacloprid (*P > 0.06*, all cases). On average, the addition of a third insecticide resulted in a 62.9 ± 13.0 % reduction in average density. Density was more greatly reduced in some groups than others with scrapers the most affected (-111.6 ± 16.9 %) and taxa richness the

**Figure 7.** Comparison of % reduction in metrics due to treatment with the ternary mixture of 0.1 TU versus the binary mixture with the same doses. Each 0.1 TU dose should reduce the density of sensitive taxa by 5% because 1 TU = LC50. Therefore, reductions greater than 5% in the density of aquatic taxa is of biological interest even if differences in the

All of the metrics of benthic invertebrate responses measured also had significant interaction terms (TU x I, *P <* 0.1) suggesting that not all taxa, groups or guilds were equally sensitive to insecticide treatment. Differential toxicity within the organophosphorus insecticides has been reported previously and is predominantly due to the complexity of the biochemical pathway to reach what is considered the primary target, acetylcholinesterase [8,9,10]. Specif‐ ically, the toxic potency of organophosphorus insecticides depends on the creation of an

density of organisms were not found to be statistically significant.

**4.1. Responses to chlorpyrifos and dimethoate**

**4. Discussion**

Despite the equivalent toxic unit doses employed in this study, treatment with dimethoate was associated with increased abundance of different taxa and guilds with the exception of predators, which were found to be substantially negatively impacted by all high dose treat‐ ments. In mixture treatments, the density of taxa often fell between that of either of the two insecticides individually, or, resembled the relatively non-toxic dimethoate at 0.4 and 0.8 TU. The highly significant declines in abundance of different taxa and guilds due to chlor‐ pyrifos treatment, and the lack of similar findings due to dimethoate treatment are troubling because this study determined the appropriate doses from standard bioassays of the same genera from public databases of the published literature. For instance, according to a Nor‐ wegian study by Baekken and Aanes [5], the 96-hr LC50 for *Baetis rhodani* exposed to dime‐ thoate was ~ 7 µg/L. In this study *Baetis* not only survived but emerged as adults (37 females and 26 males, not shown) in the 0.8 TU treatment where the dimethoate concentration was in the range of 19.93 – 22.96 µg/L. Disparities such as these invite speculation. If regional dif‐ ferences in sensitivity are as pronounced as the above finding suggests, then modeling may be restricted to more local scales. Alternatively, regional variation in data quality also invites speculation.

This study generally found that the mixture pattern at high doses had intermediate toxicity. Specifically, invertebrate responses to the binary mixtures were between that of dimethoate or chlorpyrifos individually. LeBlanc *et al.* [14] also found mixtures of chlorpyrifos and di‐ methoate to exhibit dose-level dependency in concurrent laboratory studies using chlorpyri‐ fos and dimethoate in both binary mixtures (i.e., low dose antagonism to high dose synergy). Although high dose exposures are likely less common than sublethal effects (as described in [24]), high dose synergy is a concern because isolated high-dose events (e.g., a rain event) could significantly alter the composition of aquatic communities. Additionally, in more complex mixtures where multiple modes of action may be the norm, the concentra‐ tion that initiates a synergistic effect may be lower than implied from bioassay results using single-species and single compounds.

#### **4.2. Preliminary findings for responses in binary versus ternary mixtures**

In this study, the addition of a third insecticide at 0.1 TU resulted in an average reduction in invertebrate density of approximately 60% (-62.9 ± 13.0%). However, the addition of 0.1TU of imidacloprid should, in theory, only result in a reduction of 5% in the abundance of or‐ ganisms because 0.1 TU equals the 5% median lethal concentration or the LC5. Therefore, average density was reduced 50% more with the addition of one more insecticide to the mix‐ ture despite the addition occurring at what would otherwise be considered a very low dose. The implication of these findings is that the presence of imidacloprid in a mixture, an insec‐ ticide with a similar mode of action to chlorpyrifos and dimethoate, may cause significantly greater than additive reductions in invertebrate density in naturally occuring assemblages such as those tested in this study. These findings are similar to those of Leblanc *et al.* [14] where the combined action of imidacloprid resulted in greater than additive toxicity of mix‐ tures of the same insecticides used in this study.

chemically (as in [28,29]), there appears to be little empirical evidence to support the uni‐ form toxicity, or activity of organophosphorus compounds in biota (see [9]). Rather, non-ad‐ ditive responses appear to be the norm in real systems, perhaps because effects in real systems are mediated by biotic filters such as trait-mediated indirect effects [30,31]. We sug‐ gest that grouping these compounds into potency subclasses, as first suggested by Mileson *et al.* [23] will aid modelling efforts to overcome dose dependent effects of similar mixtures with variable potency. This is particularly warranted because dose-dependency appears to be a common mixture pattern [32]. Although concentration addition is widely thought to be a conservative approach to modelling impacts in streams (as in [33]), regional differences in sensitivity, or alternatively data quality, will reduce the usefulness of additive models. Fi‐ nally, current toxicological models such as concentration addition and independent action, do not consider biological interactions between species. Interactions between species in a community can increase or mask organismal responses to stress and may be more important

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than isolated laboratory responses for the prediction of community level patterns.

predictions derived from laboratory based mode of action models.

In this study, when chlorpyrifos and dimethoate were both applied these mixtures were of‐ ten intermediately toxic to aquatic invertebrates with the exception of predators that were severely impacted by all elevated insecticide treatments. In contrast, ternary mixtures were generally more toxic than expected and predators were highly affected even at the very low doses tested. Although only an additional 0.1 TU (= LC5) was added of a third insecticide, imidacloprid, responses in the density of different benthic macroinvertebrate metrics were reduced on average by more than 20%. From a community standpoint, it is apparent that different taxa and guilds within the macroinvertebrate community tested were not equally sensitive to treatment with different insecticides despite the use of equivalent toxic unit doses drawn from published bioassays on the same genera of aquatic insects as those exam‐ ined in this study. As such, additive assumptions of toxicity in a community context are questionable. This is particularly true given that the interactions between species are rarely measured in ecotoxicology and thus, significant biological effects are likely ignored. Pest managers and regulators concerned with the impact of complex mixtures on naturally oc‐ curring communities may be better informed by focused study of common mixtures of mul‐ tiple compounds on locally and regionally relevant assemblages of organisms than

Many thanks to Kristie Heard (Environment Canada, Fredericton) for her assistance with the taxonomy and subsampling procedure, to Dave Hryn (Environment Canada, Frederic‐ ton) for his technical expertise and assistance in conducting the artificial stream experiment, and to Jon Bailey (Environment Canada, Saskatoon) who conducted the chemical analyses.

**5. Conclusions**

**Acknowledgements**

Although we did not detect significant differences when comparing the density of predators in low dose binary versus ternary mixtures, responses in groups such as predators continue to be of interest because of the importance of certain feeding groups in food webs (e.g., see [25]). For predators, the average percent reduction in density was -27.4 ± 9.9% at a dose that in theory will cause a 15% reduction in density (0.3 TU = LC15). However, if the addition of one insecticide can cause (at best) a 30% reduction in density, then what effects are likely for more complex mixtures acting on highly interconnected aquatic communities? Gilliom has previously reported that mixtures of up to 5 insecticides are routinely found in the environ‐ ment [1]. If the patterns found in this study are true of more complex mixtures, then 5 insec‐ ticides at 0.1 TU could remove more than half the invertebrate population (> LC50) at individual doses that are thought to cause a mere 5% reduction in density. Clearly, further study of the effects of mixtures on keystone species, such as predators, will be important for untangling community responses to multiple stressors.

#### **4.3. Implications to additive models: a biological argument**

It is questionable whether additive predictions of responses can be made for these insecti‐ cides despite having the same (or similar) primary modes of action. Clearly, chlorpyrifos and dimethoate were not sufficiently similar in their actions on organisms in the community assemblage studied here to warrant additive treatment, even though their effects may be similar *in vitro*. In this study, dose-level dependency and genus or guild specific differences were the norm. Therefore, although the use of additivity to predict effects of insecticide mix‐ tures has the appeal of simplicity, pest managers and regulators may be better informed by focused study of common mixtures of multiple compounds on relevant assemblages of or‐ ganisms. Differences in sensitivity and tolerance may be region or system specific due to the predisposition of different populations to up or down-regulate the production of alternative substrates to which these insecticides can bind [9,26,27].

Thus, arbitrary grouping of two similar insecticides based on their primary mode of action, is inappropriate, particularly in an ecological context. Although grouping organophospho‐ rus insecticides to model responses additively has been demonstrated to be appropriate chemically (as in [28,29]), there appears to be little empirical evidence to support the uni‐ form toxicity, or activity of organophosphorus compounds in biota (see [9]). Rather, non-ad‐ ditive responses appear to be the norm in real systems, perhaps because effects in real systems are mediated by biotic filters such as trait-mediated indirect effects [30,31]. We sug‐ gest that grouping these compounds into potency subclasses, as first suggested by Mileson *et al.* [23] will aid modelling efforts to overcome dose dependent effects of similar mixtures with variable potency. This is particularly warranted because dose-dependency appears to be a common mixture pattern [32]. Although concentration addition is widely thought to be a conservative approach to modelling impacts in streams (as in [33]), regional differences in sensitivity, or alternatively data quality, will reduce the usefulness of additive models. Fi‐ nally, current toxicological models such as concentration addition and independent action, do not consider biological interactions between species. Interactions between species in a community can increase or mask organismal responses to stress and may be more important than isolated laboratory responses for the prediction of community level patterns.
