**1. Introduction**

In this study two questions will be posed: firstly, how can single-species, single-compound toxicity test data on non-target aquatic insects predict patterns in stream communities ex‐ posed to the same compounds individually and jointly? Secondly, can mixtures of two or three insecticides be treated additively using a concentration addition, Toxic Unit (TU) ap‐ proach in an aquatic community context? To evaluate these questions, the following studies examined the responses of field-collected benthic (bottom-dwelling) invertebrates exposed to mixtures of organophosphorus insecticides (chlorpyrifos and dimethoate) in detail as well as a preliminary investigation of the effects of adding a third insecticide to the mixture, the neo-nicotinoid (imidacloprid).

Non- target aquatic organisms are routinely exposed to pesticides because these compounds are widely used and are regularly detected during stream biomonitoring [1]. Mixtures of in‐ secticides are particularly worrisome because these compounds can directly alter the abun‐ dance and diversity of aquatic insects; consequently, these effects can reshape aquatic food webs. Organophosphorus insecticides are particularly relevant for consideration because they are extensively used in agriculture worldwide and, for example, constitute ~ 40% of the insecticides applied in the United States [2]. In this study, two organophosphorus insecti‐ cides were selected, chlorpyrifos (O,O-diethyl O-(3,5,6-trichloro-2-pyridinyl) phosphoro‐ thioate) and dimethoate (O,O-dimethyl S-[2-(methylamino)-2-oxoethyl] phosphorodithioate) to examine in detail because both are among the most commonly used in North America. Both are also routinely applied jointly or sequentially for the protection of more than 40 crops globally [2,3].

Chlorpyrifos and dimethoate are also highly toxic to non-target, aquatic species. According to van Wijngaarden *et al.* [4], the 48-h LC50 (median lethal concentration to affect 50% of the

© 2013 Alexander and Culp; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Alexander and Culp; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

population) for chlorpyrifos on the non-target mayfly, *Cloeon dipterum* is approximately 1 µg/L and similarly, Baekken and Aanes [5], report that the 96-hr LC50 for the mayfly, *Baetis rhodani*, is in the range of 7 µg/L for dimethoate. The third insecticide, imidacloprid (1-((6- Chloro-3-pyridinyl)methyl)-N-nitro-2-imidazolidinimine), is also highly toxic to non-target aquatic species (e.g., the mayfly, *Epeorus longimanus* 24-h LC50 = 2.1 ± 0.5 µg/L, see [6]). Un‐ like chlorpyrifos and dimethoate however, the primary mode of action of imidacloprid is semi-permanent binding to the acetylcholine receptor rather than the ACh enzyme [7]. This difference may increase toxicity of the ternary mixture because all three insecticides bind the same enzyme and receptor system.

tebrates were collected in the Nashwaak River (sampling location: 46º14294´N, 66º36722´W). The Nashwaak River is a relatively pristine tributary of the larger Saint John River and runs more than 100 km through forested and rural communities of less than 500 inhabitants in

Subsampled invertebrate assemblages were inoculated into 88 outdoor, artificial streams (Figure 1, see also [11,12]). Each partial flow-through stream was circular and had a planar

(nreplicates per treatment = 8) were examined in detail: chlorpyrifos (control, 0.2, 0.4 and 0.8 TU), di‐ methoate (control, 0.2, 0.4 and 0.8 TU) and a 1:1 mixture of both insecticides (0.1 + 0.1, 0.2 + 0.2 and 0.4 + 0.4 TU). An additional ternary 1:1:1 mixture of all three insecticides was also examined as a pilot study and included imidacloprid as well as chlorpyrifos and dimethoate (0.1 + 0.1 + 0.1 TU). Treatment solutions were housed in polyethylene reservoirs and mani‐ folds were used to distribute the treatment solutions at uniform flow rates to each replicate stream. Groundwater from the extensive Saint John River aquifer was used to provide water to the artificial streams. Wastewater from each stream was passed through carbon filters (Culligan Inc.; activated carbon filter cylinder, Moncton, NB, CAN) to remove all contami‐

**Figure 1.** Cylindrical artificial streams. We inoculated 88 outdoor, artificial streams with a field-collected benthic inver‐ tebrate assemblage. Each flow-through stream was circular with a planar area of 0.065 m2 and a 10-L volume. In Fig. 1a, 8 streams were inoculated with gravel (coarse and fine) as well as 5 cobbles per stream. Protruding from the centre of each replicate stream is a motorized, rotating paddle that regulated the velocity of water in each stream. In Fig. 1b, streams post inoculation where each stream is covered with mesh to facilitate the collection of adult emergent insects.

Prior to initiating the experiment, benthic substrates were introduced into each replicate stream. A realistic benthic substrate was created by inoculating each stream with a mix‐

nants before any water was discharged to the environment.

**2.1. Establishment of the aquatic community**

*2.1.1. Mimicking in-stream habitats*

and a 10-L volume. Three treatments of organophosphorus insecticides

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85

central New Brunswick.

area of 0.065 m2

Organophosphorus insecticides are thought to primarily target the acetylcholinesterase (AChE) enzyme, preventing the removal of acetylcholine (ACh) by the enzyme from the post-synaptic gap [8]. Therefore, excessive acetylcholine is bound and continuous nerve signals are sent to cholinergic receptors, which can result in trembling, respiratory duress and ultimately death [8]. Notably, in order for most organophosphorus compounds to become toxic they must first be transformed into their active form, an oxon [9,10]. How‐ ever, insecticides such as chlorpyrifos and dimethoate are chemically diverse and are able to interact with multiple metabolic pathways and targets. Therefore, indirect bio‐ chemical or ecological effects of these compounds may be responsible for observed dif‐ ferences in their toxicity [8,9,10].

In this study, two organophosphorous insecticides (chlorpyrifos and dimethoate) with the same primary mode of action were tested individually and jointly on a natural, macroinver‐ tebrate assemblage using a toxic unit approach. The primary question asked was whether the joint-action of these two insecticides can be reasonably evaluated at a community level using additive assumptions of toxicity. This question was evaluated by determining the ap‐ propriate concentrations in toxic units of chlorpyrifos and dimethoate by compiling singlespecies toxicity test data for orders of insects commonly thought to be sensitive indicators in aquatic biomonitoring of streams and rivers namely, Ephemeroptera, Plecoptera and Tri‐ choptera, or E.P.T. taxa. A 20 day artificial stream experiment was conducted where fieldcollected benthic (bottom-dwelling) macroinvertebrate assemblages were exposed to four toxic unit (TU) doses of either chlorpyrifos or dimethoate individually (control, 0.2, 0.4 and 0.8 TU) and two, 1:1 mixture doses (0.2 + 0.2 TU and 0.4 + 0.4 TU) of both insecticides ap‐ plied jointly. Subsequently, responses in the benthos in a community were examined using Principle Components Analysis (PCA). Macroinvertebrate abundance, richness and guild structure was assessed using a factorial ANOVA and a chi-square (χ<sup>2</sup> ) approach to compare observed responses to control values as well as to predicted responses to treatment across a toxic unit gradient.

### **2. Methods**

This 20-d study was conducted from 12 July to 2 August, 2007 at the Environment Canada mesocosm facility 10-km southeast of Fredericton (New Brunswick, Canada). Aquatic inver‐ tebrates were collected in the Nashwaak River (sampling location: 46º14294´N, 66º36722´W). The Nashwaak River is a relatively pristine tributary of the larger Saint John River and runs more than 100 km through forested and rural communities of less than 500 inhabitants in central New Brunswick.

Subsampled invertebrate assemblages were inoculated into 88 outdoor, artificial streams (Figure 1, see also [11,12]). Each partial flow-through stream was circular and had a planar area of 0.065 m2 and a 10-L volume. Three treatments of organophosphorus insecticides (nreplicates per treatment = 8) were examined in detail: chlorpyrifos (control, 0.2, 0.4 and 0.8 TU), di‐ methoate (control, 0.2, 0.4 and 0.8 TU) and a 1:1 mixture of both insecticides (0.1 + 0.1, 0.2 + 0.2 and 0.4 + 0.4 TU). An additional ternary 1:1:1 mixture of all three insecticides was also examined as a pilot study and included imidacloprid as well as chlorpyrifos and dimethoate (0.1 + 0.1 + 0.1 TU). Treatment solutions were housed in polyethylene reservoirs and mani‐ folds were used to distribute the treatment solutions at uniform flow rates to each replicate stream. Groundwater from the extensive Saint John River aquifer was used to provide water to the artificial streams. Wastewater from each stream was passed through carbon filters (Culligan Inc.; activated carbon filter cylinder, Moncton, NB, CAN) to remove all contami‐ nants before any water was discharged to the environment.

**Figure 1.** Cylindrical artificial streams. We inoculated 88 outdoor, artificial streams with a field-collected benthic inver‐ tebrate assemblage. Each flow-through stream was circular with a planar area of 0.065 m2 and a 10-L volume. In Fig. 1a, 8 streams were inoculated with gravel (coarse and fine) as well as 5 cobbles per stream. Protruding from the centre of each replicate stream is a motorized, rotating paddle that regulated the velocity of water in each stream. In Fig. 1b, streams post inoculation where each stream is covered with mesh to facilitate the collection of adult emergent insects.

#### **2.1. Establishment of the aquatic community**

#### *2.1.1. Mimicking in-stream habitats*

population) for chlorpyrifos on the non-target mayfly, *Cloeon dipterum* is approximately 1 µg/L and similarly, Baekken and Aanes [5], report that the 96-hr LC50 for the mayfly, *Baetis rhodani*, is in the range of 7 µg/L for dimethoate. The third insecticide, imidacloprid (1-((6- Chloro-3-pyridinyl)methyl)-N-nitro-2-imidazolidinimine), is also highly toxic to non-target aquatic species (e.g., the mayfly, *Epeorus longimanus* 24-h LC50 = 2.1 ± 0.5 µg/L, see [6]). Un‐ like chlorpyrifos and dimethoate however, the primary mode of action of imidacloprid is semi-permanent binding to the acetylcholine receptor rather than the ACh enzyme [7]. This difference may increase toxicity of the ternary mixture because all three insecticides bind the

Organophosphorus insecticides are thought to primarily target the acetylcholinesterase (AChE) enzyme, preventing the removal of acetylcholine (ACh) by the enzyme from the post-synaptic gap [8]. Therefore, excessive acetylcholine is bound and continuous nerve signals are sent to cholinergic receptors, which can result in trembling, respiratory duress and ultimately death [8]. Notably, in order for most organophosphorus compounds to become toxic they must first be transformed into their active form, an oxon [9,10]. How‐ ever, insecticides such as chlorpyrifos and dimethoate are chemically diverse and are able to interact with multiple metabolic pathways and targets. Therefore, indirect bio‐ chemical or ecological effects of these compounds may be responsible for observed dif‐

In this study, two organophosphorous insecticides (chlorpyrifos and dimethoate) with the same primary mode of action were tested individually and jointly on a natural, macroinver‐ tebrate assemblage using a toxic unit approach. The primary question asked was whether the joint-action of these two insecticides can be reasonably evaluated at a community level using additive assumptions of toxicity. This question was evaluated by determining the ap‐ propriate concentrations in toxic units of chlorpyrifos and dimethoate by compiling singlespecies toxicity test data for orders of insects commonly thought to be sensitive indicators in aquatic biomonitoring of streams and rivers namely, Ephemeroptera, Plecoptera and Tri‐ choptera, or E.P.T. taxa. A 20 day artificial stream experiment was conducted where fieldcollected benthic (bottom-dwelling) macroinvertebrate assemblages were exposed to four toxic unit (TU) doses of either chlorpyrifos or dimethoate individually (control, 0.2, 0.4 and 0.8 TU) and two, 1:1 mixture doses (0.2 + 0.2 TU and 0.4 + 0.4 TU) of both insecticides ap‐ plied jointly. Subsequently, responses in the benthos in a community were examined using Principle Components Analysis (PCA). Macroinvertebrate abundance, richness and guild

observed responses to control values as well as to predicted responses to treatment across a

This 20-d study was conducted from 12 July to 2 August, 2007 at the Environment Canada mesocosm facility 10-km southeast of Fredericton (New Brunswick, Canada). Aquatic inver‐

) approach to compare

structure was assessed using a factorial ANOVA and a chi-square (χ<sup>2</sup>

same enzyme and receptor system.

84 Insecticides - Development of Safer and More Effective Technologies

ferences in their toxicity [8,9,10].

toxic unit gradient.

**2. Methods**

Prior to initiating the experiment, benthic substrates were introduced into each replicate stream. A realistic benthic substrate was created by inoculating each stream with a mix‐ ture of 25% fine gravel (2 - 4 mm) and 75% gravel (4 - 30 mm) that was obtained from gravel beds adjacent to the invertebrate sampling site on the Nashwaak River (Figure 1a). Cobblestones (7-10 cm) were also collected from this site with five stones randomly assigned to each replicate stream. Cobble and gravel were gently washed to remove any attached invertebrates while maintaining the periphyton community. This procedure es‐ tablished a lotic substrate consisting of a 2-3 cm layer of gravel-cobble plus surface stones that were covered with periphyton and was similar to the original habitat of the benthic community examined (Figure 1a).

ucts Canada Inc., Dorchester, ON, Canada) and finally, imidacloprid (240 µg/L) by dilution of Admire 240® (Bayer CropScience, Calgary, AB, CAN). The insecticide-treated groundwa‐ ter was delivered to one of eleven treatment reservoirs by positive displacement pumps (Viking Pumps, Pulsefeeder 25-H duplex pump, Cedar Falls, IA, USA). Secondary pumps then delivered the treatment solutions from each reservoir through a manifold to generate

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**Figure 2.** Benthic community subsampling and inoculation procedure for 88 replicate streams (11 treatments each containing 8 replicates). Sets of 25 U-nets (5 samplers collecting 5 U-nets each) were subsampled into 16 equal parts using a pie-plate made from 44 µm mesh. One sixteenth (1/16) of every 20 U-nets collected was inoculated into one replicate stream in every treatment level. This procedure was repeated eight times with each additional set of 25 Unets systematically inoculated into adjacent replicate streams (one per treatment level). Thus, if the initial stream com‐ munity had been significantly different in composition differences would have been allocated between treatments. Differences in community composition were not detected between subsamples (*Wilks-L >* 0.86; *P >* 0.99, in both cas‐

es).

uniform flow rates into the base of each partial flow-through, replicate stream.

#### *2.1.2. Field collection*

Benthic invertebrates were collected in a single riffle upstream of the gravel collection site on the Nashwaak River with U-nets (area = 0.06 m2 ). The subsampling procedure consisted of the collection of twenty-five (25) U-nets collected 8 times by 5 samplers working system‐ atically upstream within the riffle. Twenty-five U-nets were selected to slightly increase (~10%) the ambient density of aquatic invertebrates in the artificial streams, thus offsetting any mortality due to transport from the river to the test site. Each set of 25 U-nets were div‐ ided into 16 community subsamples with 5 reference subsamples from each set retained to determine the initial composition of the aquatic community. Streams were systematically in‐ oculated with a subsample from each of the 8 sets of the 25 U-nets collected. Such that each of the 11 treatments levels (low, medium, high or chlorpyrifos, dimethoate, binary mixture, as well as a single comparison of a low ternary mixture and the control) received a portion of the same stream assemblages collected in the field (Figure 2).

#### **2.2. Establishment of treatments**

The 96-h LC50s (as 95% C.I.) were estimated for chlorpyrifos (4.68 – 5.69 µg/L) and dime‐ thoate (23.96 – 26.57 µg/L) by curve-fitting single-species, single-compound toxicity test data compiled from public databases (U.S. Environmental Protection Agency Ecotox database [13], Figure 3). Appropriateness of doses was also assessed using tandem laboratory testing of chlorpyrifos and dimethoate on laboratory-reared *Chironomus tentans* and field-collected Heptageniidae mayflies from the Nashwaak River [14]. For imidacloprid (96-h LC50 0.8 – 3.1 µg/L 95% C.I.), where less data was available, appropriate doses were determined in com‐ parison to previous artificial stream studies in our region [15]. Only genera of the orders Ephemeroptera, Plecoptera and Trichoptera (E.P.T. taxa) were included in the estimated riv‐ erine community 96-h LC50 (the median lethal concentration that will affect 50% of E.P.T. taxa) because the abundance of these insects is generally thought to be indicative of healthy streams and is widely used in stream biomonitoring [16].

Insecticide solutions were mixed in agricultural grade stock tanks, a 2000-L stock tank for chlorpyrifos, a 520-L stock tank for dimethoate and a 200–L stock tank of each component of the ternary mixture. All solutions were mixed using groundwater from the extensive Saint John River aquifer. Stock solutions of chlorpyrifos (70 µg/L) were made by serial dilution of Lorsban -4E© (NAF-163, Dow AgroSciences, Indianapolis, IN, USA). Stock solutions of di‐ methoate (200 µg/L) were made by serial dilution of Lagon 480E © (9382, United Agri Prod‐ ucts Canada Inc., Dorchester, ON, Canada) and finally, imidacloprid (240 µg/L) by dilution of Admire 240® (Bayer CropScience, Calgary, AB, CAN). The insecticide-treated groundwa‐ ter was delivered to one of eleven treatment reservoirs by positive displacement pumps (Viking Pumps, Pulsefeeder 25-H duplex pump, Cedar Falls, IA, USA). Secondary pumps then delivered the treatment solutions from each reservoir through a manifold to generate uniform flow rates into the base of each partial flow-through, replicate stream.

ture of 25% fine gravel (2 - 4 mm) and 75% gravel (4 - 30 mm) that was obtained from gravel beds adjacent to the invertebrate sampling site on the Nashwaak River (Figure 1a). Cobblestones (7-10 cm) were also collected from this site with five stones randomly assigned to each replicate stream. Cobble and gravel were gently washed to remove any attached invertebrates while maintaining the periphyton community. This procedure es‐ tablished a lotic substrate consisting of a 2-3 cm layer of gravel-cobble plus surface stones that were covered with periphyton and was similar to the original habitat of the

Benthic invertebrates were collected in a single riffle upstream of the gravel collection site

of the collection of twenty-five (25) U-nets collected 8 times by 5 samplers working system‐ atically upstream within the riffle. Twenty-five U-nets were selected to slightly increase (~10%) the ambient density of aquatic invertebrates in the artificial streams, thus offsetting any mortality due to transport from the river to the test site. Each set of 25 U-nets were div‐ ided into 16 community subsamples with 5 reference subsamples from each set retained to determine the initial composition of the aquatic community. Streams were systematically in‐ oculated with a subsample from each of the 8 sets of the 25 U-nets collected. Such that each of the 11 treatments levels (low, medium, high or chlorpyrifos, dimethoate, binary mixture, as well as a single comparison of a low ternary mixture and the control) received a portion

The 96-h LC50s (as 95% C.I.) were estimated for chlorpyrifos (4.68 – 5.69 µg/L) and dime‐ thoate (23.96 – 26.57 µg/L) by curve-fitting single-species, single-compound toxicity test data compiled from public databases (U.S. Environmental Protection Agency Ecotox database [13], Figure 3). Appropriateness of doses was also assessed using tandem laboratory testing of chlorpyrifos and dimethoate on laboratory-reared *Chironomus tentans* and field-collected Heptageniidae mayflies from the Nashwaak River [14]. For imidacloprid (96-h LC50 0.8 – 3.1 µg/L 95% C.I.), where less data was available, appropriate doses were determined in com‐ parison to previous artificial stream studies in our region [15]. Only genera of the orders Ephemeroptera, Plecoptera and Trichoptera (E.P.T. taxa) were included in the estimated riv‐ erine community 96-h LC50 (the median lethal concentration that will affect 50% of E.P.T. taxa) because the abundance of these insects is generally thought to be indicative of healthy

Insecticide solutions were mixed in agricultural grade stock tanks, a 2000-L stock tank for chlorpyrifos, a 520-L stock tank for dimethoate and a 200–L stock tank of each component of the ternary mixture. All solutions were mixed using groundwater from the extensive Saint John River aquifer. Stock solutions of chlorpyrifos (70 µg/L) were made by serial dilution of Lorsban -4E© (NAF-163, Dow AgroSciences, Indianapolis, IN, USA). Stock solutions of di‐ methoate (200 µg/L) were made by serial dilution of Lagon 480E © (9382, United Agri Prod‐

). The subsampling procedure consisted

benthic community examined (Figure 1a).

86 Insecticides - Development of Safer and More Effective Technologies

**2.2. Establishment of treatments**

on the Nashwaak River with U-nets (area = 0.06 m2

of the same stream assemblages collected in the field (Figure 2).

streams and is widely used in stream biomonitoring [16].

*2.1.2. Field collection*

**Figure 2.** Benthic community subsampling and inoculation procedure for 88 replicate streams (11 treatments each containing 8 replicates). Sets of 25 U-nets (5 samplers collecting 5 U-nets each) were subsampled into 16 equal parts using a pie-plate made from 44 µm mesh. One sixteenth (1/16) of every 20 U-nets collected was inoculated into one replicate stream in every treatment level. This procedure was repeated eight times with each additional set of 25 Unets systematically inoculated into adjacent replicate streams (one per treatment level). Thus, if the initial stream com‐ munity had been significantly different in composition differences would have been allocated between treatments. Differences in community composition were not detected between subsamples (*Wilks-L >* 0.86; *P >* 0.99, in both cas‐ es).

**Treatment in Toxic Units (TU) 0.2 TU 0.4 TU 0.8 TU**

**Mixtures in Toxic Units (TU x n) 0.1 TU x 2 0.2 TU x 2 0.4 TU x 2 0.1 TU x 3**

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Target [chlorpyrifos] 0.24 - 0.57 0.94 – 1.14 1.87 – 2.28 0.24 - 0.57 Actual [chlorpyrifos] 0.19 - 0.86 0.78 – 1.61 1.39 – 4.02 0.12 - 0.38 Target [dimethoate] 2.40 - 2.66 4.79 – 5.31 9.58 – 10.63 2.40 - 2.66 Actual [dimethoate] 2.13 – 3.54 2.36 – 5.88 8.18 – 16.43 2.18 - 2.80 Target [imidacloprid] 0.24 - 0.57 Actual [imidacloprid] 0.47 - 0.69

**Table 1.** Comparison of treatments in toxic units (TU) with respect to the 95% confidence interval (95% CI) of the estimated range of targeted doses and the actual concentrations for chlorpyrifos, dimethoate and the 1:1 binary (x2) mixtures of chlorpyrifos and dimethoate compared to 1:1:1 ternary (x3) insecticide mixtures of chlorpyrifos, dimethoate and imidacloprid. All concentrations are in µg/L. Target concentrations for each insecticide are presented

At the end of the 20-d experiment, the streams were dismantled and the contents collected. Water samples, periphyton samples and invertebrates were collected from each replicate stream. Benthic macroinvertebrates were collected from each stream and preserved (10% formalin, transferred to 70% ethanol after 1 week) for subsequent laboratory sorting and identification using dissecting microscopes (Leica© Microsystems Ltd., Cambridge, UK). Aquatic specimens were sorted and identified to genus at the end of the experiment accord‐ ing to Environment Canada protocols, with a minimum of 20% of the collected material checked by a certified taxonomist to achieve 95% confidence in the identifications [17]. Some taxa were only identified to Order given time constraints and available expertise (e.g., Oli‐ gochaeta, Nematoda, Gastropoda, Collembola and 1st instar Plecoptera). Guilds were infer‐ red from the literature in order to infer the habits of organisms [16,18]. Adult insects were also collected over the course of the 20-d experiment in 2-d intervals and in some cases were

Community responses were examined in the factorial portion of the experiment (chlorpyri‐ fos x dimethoate) using Principal Components Analysis (PCA) because the data were con‐ tinuous with respect to both of the treatment level factors of interest (e.g., actual

Target [chlorpyrifos] 0.94 – 1.14 1.87 – 2.28 3.74 – 4.55 Actual [chlorpyrifos] 0.47 – 1.31 1.64 – 2.70 2.41 – 6.89 Target [dimethoate] 3.79 – 5.31 9.58 – 10.63 19.17 – 21.26 Actual [dimethoate] 1.04 – 4.80 9.32 – 12.07 19.93 – 22.96

Target [imidacloprid] N/A N/A N/A Actual [imidacloprid] N/A N/A N/A

as ranges to reflect the uncertainty in the LC50 estimate.

used to corroborate the presence of cryptic genera.

**2.3. Final data collection**

**2.4. Statistical approaches**

**Figure 3.** Percent Affected (96-h) of E.P.T. taxa as reported in the literature for the insecticides chlorpyrifos and dime‐ thoate. For imidacloprid (96-h LC50 0.8 – 3.1 µg/L 95% C.I.), where less data was available, appropriate doses were determined in comparison to previous studies in our region [6,15]. Additional, tandem laboratory testing of chlorpyri‐ fos and dimethoate on laboratory-reared *Chironomus tentans* and field-collected Heptageniidae mayflies from the Nashwaak River further corroborated dose selection [14]. Only genera of the E.P.T. Orders (Ephemeroptera, Plecoptera and Trichoptera) were used because the abundance of these insects is thought to be indicative of healthy stream con‐ ditions.

Chemical analysis determined the actual concentrations (Table 1) of the three insecticides in‐ dividually and in mixture. Analyses were conducted at the National Water Research Insti‐ tute (Environment Canada) in Saskatoon (SK, Canada) using a Waters 2695 Alliance HPLC System interfaced to a Micromass Quattro Ultima triple quadrupole mass spectrometer (LC-MS-MS) equipped with an electrospray ionization interface set to positive ion mode. For chlorpyrifos and dimethoate, chromatography was achieved using a Waters Xtera MS C18 (100 mm x 2.1 mm i.d., 3.5-µm particle size, Milford, MA, USA) analytical column and an aqueous acetonitrile mobile phase containing 0.1% formic acid (v/v). For imidacloprid, the mobile phase contained 40% aqueous acetonitrile and 0.2% formic acid (v/v). Water samples were collected in each treatment level on three occasions (July 13, 14, 17 in 2007) during the 96-h insecticide exposure period which began at noon on 13 July. Samples were collected in 500-mL amber vials (EPA vials, Fisher scientific, Fair Lawn, NJ, USA) and stored at 4ºC until shipment to Saskatoon for analysis. The samples were subjected to solid-phase (dimethoate) or liquid-phase (chlorpyrifos) extraction, the extracts taken to dryness, and the extract resi‐ due dissolved in deionized water (1.0 mL) prior to analysis by LC-MS-MS. All of the actual concentrations overlapped the target concentrations (Table 1) with an even distribution of under- and over- dosing for each target. Therefore, concentrations were comparable to those determined by laboratory bioassays in the published literature.


**Table 1.** Comparison of treatments in toxic units (TU) with respect to the 95% confidence interval (95% CI) of the estimated range of targeted doses and the actual concentrations for chlorpyrifos, dimethoate and the 1:1 binary (x2) mixtures of chlorpyrifos and dimethoate compared to 1:1:1 ternary (x3) insecticide mixtures of chlorpyrifos, dimethoate and imidacloprid. All concentrations are in µg/L. Target concentrations for each insecticide are presented as ranges to reflect the uncertainty in the LC50 estimate.

#### **2.3. Final data collection**

**Figure 3.** Percent Affected (96-h) of E.P.T. taxa as reported in the literature for the insecticides chlorpyrifos and dime‐ thoate. For imidacloprid (96-h LC50 0.8 – 3.1 µg/L 95% C.I.), where less data was available, appropriate doses were determined in comparison to previous studies in our region [6,15]. Additional, tandem laboratory testing of chlorpyri‐ fos and dimethoate on laboratory-reared *Chironomus tentans* and field-collected Heptageniidae mayflies from the Nashwaak River further corroborated dose selection [14]. Only genera of the E.P.T. Orders (Ephemeroptera, Plecoptera and Trichoptera) were used because the abundance of these insects is thought to be indicative of healthy stream con‐

88 Insecticides - Development of Safer and More Effective Technologies

Chemical analysis determined the actual concentrations (Table 1) of the three insecticides in‐ dividually and in mixture. Analyses were conducted at the National Water Research Insti‐ tute (Environment Canada) in Saskatoon (SK, Canada) using a Waters 2695 Alliance HPLC System interfaced to a Micromass Quattro Ultima triple quadrupole mass spectrometer (LC-MS-MS) equipped with an electrospray ionization interface set to positive ion mode. For chlorpyrifos and dimethoate, chromatography was achieved using a Waters Xtera MS C18 (100 mm x 2.1 mm i.d., 3.5-µm particle size, Milford, MA, USA) analytical column and an aqueous acetonitrile mobile phase containing 0.1% formic acid (v/v). For imidacloprid, the mobile phase contained 40% aqueous acetonitrile and 0.2% formic acid (v/v). Water samples were collected in each treatment level on three occasions (July 13, 14, 17 in 2007) during the 96-h insecticide exposure period which began at noon on 13 July. Samples were collected in 500-mL amber vials (EPA vials, Fisher scientific, Fair Lawn, NJ, USA) and stored at 4ºC until shipment to Saskatoon for analysis. The samples were subjected to solid-phase (dimethoate) or liquid-phase (chlorpyrifos) extraction, the extracts taken to dryness, and the extract resi‐ due dissolved in deionized water (1.0 mL) prior to analysis by LC-MS-MS. All of the actual concentrations overlapped the target concentrations (Table 1) with an even distribution of under- and over- dosing for each target. Therefore, concentrations were comparable to those

determined by laboratory bioassays in the published literature.

ditions.

At the end of the 20-d experiment, the streams were dismantled and the contents collected. Water samples, periphyton samples and invertebrates were collected from each replicate stream. Benthic macroinvertebrates were collected from each stream and preserved (10% formalin, transferred to 70% ethanol after 1 week) for subsequent laboratory sorting and identification using dissecting microscopes (Leica© Microsystems Ltd., Cambridge, UK). Aquatic specimens were sorted and identified to genus at the end of the experiment accord‐ ing to Environment Canada protocols, with a minimum of 20% of the collected material checked by a certified taxonomist to achieve 95% confidence in the identifications [17]. Some taxa were only identified to Order given time constraints and available expertise (e.g., Oli‐ gochaeta, Nematoda, Gastropoda, Collembola and 1st instar Plecoptera). Guilds were infer‐ red from the literature in order to infer the habits of organisms [16,18]. Adult insects were also collected over the course of the 20-d experiment in 2-d intervals and in some cases were used to corroborate the presence of cryptic genera.

#### **2.4. Statistical approaches**

Community responses were examined in the factorial portion of the experiment (chlorpyri‐ fos x dimethoate) using Principal Components Analysis (PCA) because the data were con‐ tinuous with respect to both of the treatment level factors of interest (e.g., actual 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‐ OVA (for chlorpyrifos and dimethoate only) and chi-square (χ<sup>2</sup> ) approaches. In this study, 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‐ ences to those expected (or predicted) using chi-square (χ<sup>2</sup> ) tests. Expected values were 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 density per cm2 were tested for significance, the responses are shown as the percent reduc‐ tion in response between the ternary and the binary mixtures at 0.1 TU.

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‐

Predicting the Effects of Insecticide Mixtures on Non-Target Aquatic Communities

http://dx.doi.org/10.5772/53356

91

**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

Significant change in measures of average total density per cm2 and average taxa richness

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.

(Figure 5) were only found at the highest dose of chlorpyrifos tested (0.8 TU, abun‐

orders. Remaining labels indicate genera of aquatic insect taxa (e.g., *Chironomus* spp.).

per cm2

ated with predictions of additive toxicity in toxic units (Factor 1).
