The present study examined the effects of pulse exposures of the insecticide imidacloprid on the mayfly, Epeorus longimanus Eaton (Family Heptageniidae), and on an aquatic oligochaete, Lumbriculus variegatus Müller (Family Lumbriculidae). Pulse exposures of imidacloprid are particularly relevant for examination, because this insecticide is relatively soluble (510 mg/L) and is most likely to be at effect concentrations during runoff events. Experiments examined the recovery of organisms after a 24-h pulse exposure to imidacloprid over an environmentally realistic range of concentrations (0, 0.1, 0.5, 1, 5, and 10 μg/L). Effects on feeding were measured by quantifying the algal biomass consumed by mayflies or foodstuffs egested by oligochaetes. Imidacloprid was highly toxic, with low 24-h median lethal concentrations (LC50s) in early mayfly instars (24-h LC50, 2.1 ± 0.8 μg/L) and larger, later mayfly instars (24-h LC50, 2.1 ± 0.5 μg/L; 96-h LC50, 0.65 ± 0.15 μg/L). Short (24-h) pulses of imidacloprid in excess of 1 μg/L caused feeding inhibition, whereas recovery (4 d) varied, depending on the number of days after contaminant exposure. In contrast to mayflies, oligochaetes were relatively insensitive to imidacloprid during the short (24-h) pulse; however, immobility of oligochaetes was observed during a 4-d, continuous-exposure experiment, with 96-h median effective concentrations of 6.2 ± 1.4 μg/L. Overall, imidacloprid reduced the survivorship, feeding, and egestion of mayflies and oligochaetes at concentrations greater than 0.5 but less than 10 μg/L. Inhibited feeding and egestion indicate physiological and behavioral responses to this insecticide.
The inhibition of invertebrate feeding in response to contaminant exposure can indicate the potential for a stressor to produce sublethal population-level responses . Feeding inhibition is a particularly relevant endpoint for measuring the effects of modern chemicals, such as insecticides, that are applied in low doses and that produce exposure regimes of short duration and low magnitude. One example of this new generation of chemicals is imidacloprid, a soluble (510 mg/L) insecticide commonly applied in North America . Imidacloprid is a chemically stable mimic of nicotine that has well-documented toxicity to a variety of pest species, including fleas, thrips, and the Colorado potato beetle . Because imidacloprid is relatively water soluble (510 mg/L), mobile in soil [4,5], and persistent in organic sediments (>400 d ), this compound has the potential to enter streams in concentrated pulses after rain events. In New Brunswick (Canada) and Prince Edward Island (Canada), agricultural runoff of imidacloprid has been measured over a range (mean ± standard error) of 0.25 ± 0.07 to 15.88 ± 0.99 μg/L . Also, because imidacloprid attacks the nervous system by binding to the nicotinic acetylcholine receptor (nAcChR) , a receptor common in most invertebrate taxa, it is hypothesized to cause effects in nontarget aquatic invertebrates.
Imidacloprid has been found in streams and rivers and is likely to be bioavailable to aquatic organisms. Because few studies have examined the toxicity of imidacloprid to relevant lotic species, the present study investigated the impact of environmentally relevant concentrations of imidacloprid on the feeding and egestion of two common aquatic species, the larval mayfly, Epeorus longimanus Eaton (Family Heptageniidae), and an aquatic oligochaete, Lumbriculus variegatus Müller (Family Lumbriculidae). Effects were measured using a combination of traditional toxicological (median effective concentration [EC50] and median lethal concentration [LC50]) and sublethal (feeding and egestion) endpoints to determine the impact of low-magnitude (μg/L) and short-duration (24-h) pulses of imidacloprid. We hypothesized that because imidacloprid induces tremors and lethargy in insects, the reduced activity associated with imidacloprid exposure also would reduce feeding and egestion. Furthermore, imidacloprid is not permanently bound to the nAcChR ; therefore, we also examined the latency of effects by measuring feeding rate over a 4-d recovery period.
MATERIALS AND METHODS
Organism collection and culturing
Mayfly larvae (E. longimanus) were collected in the Nash-waak River, a tributary of the larger Saint John River near Stanley (NB, Canada; 46°17.18'N, 66°44.14'W) and immediately transported to the laboratory. Epeorus longimanus are grazing mayflies that reside on the cobble substrate found in fast-flowing rivers; they were chosen for this study because mayflies are abundant in streams  and sensitive to pesticides . Larvae were equilibrated to 20 ± 1°C in Percival® (Percival Scientific, Boone, IA, USA) growth chambers over a 24-h period with a 50% water exchange to dechlorinated (model 20–36 dechlorinator; Culligan, NB, Canada) groundwater (pH, 8.1 ± 0.1; conductivity, 261 ± 5 μS). The groundwater supplied to our animal care facility at the University of New Brunswick (Fredericton, NB, Canada) is provincially monitored, and monthly reports indicated insignificant changes in water quality for the duration of our experiments. Feeding rate and lethality experiments were conducted using early instar mayflies collected throughout the spring of 2004 (body length, 3.01 ± 0.26 mm; nmeasured = 120, ntotal = 600), and later-instar larvae were collected in July of 2005 (body length, 7.5 ± 0.34 mm; nmeasured = 72, ntotal = 200). Only the later-instar mayflies were used to examine feeding recovery over time.
Oligochaetes (L. variegatus, strain 285120; Aquatic Ecosystems, Apopka, FL, USA) were cultured in the laboratory using the methods described by Williams . Oligochaetes were maintained in 10-L aquaria with dechlorinated (model 20–36 dechlorinator; Culligan) groundwater flowing through the aquaria at a rate of 200 ml/min. Oligochaetes were fed a 1:1 mixture of ground Tetramin® (Tetra, Blacksburg, VA, USA) and Spirulina spp. sinking-pellets (SP1; Aquatic Ecosystems), with 5 to 25 g provided to the oligochaete aquaria on alternate days. A 1-cm layer of shredded paper towel served as substrate. Oligochaetes chosen for lethality and egestion experiments had similar mass and length to minimize any potential confounding effects of body size . Oligochaetes selected for experiments were approximately 2.5 cm in length and had a dry mass of 1.17 ± 0.02 mg (nmeasured = 42, ntotal = 150).
Lethality and immobility tests
In tandem with feeding tests (described below), 24- and 96-h lethality and immobility tests were conducted over a range of imidacloprid concentrations (0, 0.1, 0.5, 1, 5, 10, 100, and 240 μg/L) by employing standard acute-toxicity test techniques . Mayflies and oligochaetes were exposed to aqueous solutions of imidacloprid in glass beakers (diameter, 12 cm; volume, 300 ml). Either five mayflies or 25 oligochaetes were exposed in each treatment, and each test vessel was replicated three times. Early mayfly tests were repeated several times to confirm the low LC50 result. Three 24-h LC50 tests were conducted with early instar mayflies, whereas LC50 was examined once with respect to both the 24- and 96-h exposures in later-instar mayflies. For oligochaetes, immobility was used as the endpoint to estimate the 96-h EC50 (i.e., only conducted once). Immobility was evaluated as the percentage of oligochaetes moving after gentle agitation with a transfer pipette. No immobility was detected in oligochaetes during the 24-h pulse exposure.
Mayfly feeding tests
Mayfly foodstuffs were cultured in the laboratory on ceramic tiles. Nitzschia spp. diatoms (strain F110; University of Toronto, Toronto, ON, Canada) were maintained in the exponential growth phase in S-diatom media [14,15] before being subcultured to ceramic tiles (2.5 × 2.5 cm) to form single-species diatom mats within 7 to 10 d. All cultures were raised at 20 ± 1°C under a 16:8-h light:dark photoperiod. Diatom tiles were provided ad libitum to the mayflies at a rate of four tiles/replicate stream/d.
All sublethal experiments were conducted using a 24-h pulse scenario. Experiments with early instar mayflies included five replicates of each imidacloprid concentration (0, 0.1, 0.5, 1, 5, and 10 μg/L) prepared in dechlorinated groundwater, whereas late-instar treatments were replicated 10 times to ensure that at least five replicate treatments were available for feeding rate studies. The concentrations that we chose to examine in the sublethal mayfly studies overlapped the LC50s, which were determined in tandem with our feeding studies. In the mayfly feeding experiments, each acid-washed glass beaker (diameter, 12 cm) contained four tiles covered with Nitzschia spp. and five larval mayflies collected from the field as described above. A stir bar was used to generate water velocity in the beakers to increase water flow over the external gills, therefore eliminating the need for aeration. Flow rates were maintained at 20 ml/min to prevent damage to mayflies drifting between tiles. Following 24-h exposure to the contaminant, mayflies were transferred to 300-ml, flow-through, artificial streams  that were void of imidacloprid and had four diatom-covered tiles each. Artificial streams were placed in growth chambers (Percival Scientific) and supplied with dechlorinated groundwater that recirculated through the streams in a closed system for 4 d postexposure.
Daily diatom consumption by mayflies in the postexposure streams was measured by scraping the remaining diatomaceous material from selected tiles, then washing this material onto a 47-mm, glass-fiber filter (GF-C; Whatman C grade filters; pore size, 1.2 μm; Fisher Scientific, Fair Lawn, NJ, USA). Each stream received fresh tiles daily. Preweighed filter papers were dried for 4 h at 350°C, then reweighed and the mass of diatoms remaining on the tiles after mayfly feeding determined by the difference between the initial and measured final filter mass. Feeding rate was quantified as the per-capita mass of diatoms removed from the tiles per mayfly per day (μg/mayfly/d). Because the unit of measure is 1 d, the rate is expressed as μg/mayfly throughout. Six tiles from each Nitzschia culture batch were retained, and batches were found not to be significantly different (p > 0.05). Nonconsumptive losses were measured by chlorophyll a analysis of the aqueous treatment solutions by fluorometry (lower limit, 0.05 μg/L; model 10 series; Turner Design, Sunnyvale, CA, USA). For each treatment replicate, a 10-ml aliquot of treatment water was filtered (GFC grade, as above), and chlorophyll a was extracted from the filter with 90% ethanol for 5 min in an 80°C water bath. The resulting readings (μg/L) were converted (with respect to dilution) and the resultant biomass measures subtracted from the feeding rate totals to account for nonconsumptive losses, such as careless feeding and/or sloughing of diatomaceous material because of mayfly activity within the beakers and artificial streams. In the absence of mayflies, ambient diatom sloughing was less than 0.2 ± 0.0009 μg per 300-ml test vessel.
Oligochaete egestion tests
All oligochaete egestion experiments were conducted using 24-h pulses of imidacloprid (0, 0.1, 0.5, 1, 5, and 10 μg/L) prepared in dechlorinated groundwater. Exposures were performed in glass beakers (diameter, 5 cm; volume, 80 ml), with five replicates per treatment and with each replicate containing five oligochaetes. Each beaker contained 4 g of lake sediment (organic matter, 16% ± 0.1%) capped with a fine layer of inorganic sand (Ottawa Sand Standard; 20–30 mesh; Fisher Scientific). The sand was necessary to separate the sediment and aqueous layer for subsequent fecal pipetting. Each treatment beaker also contained 60 ml of dechlorinated groundwater.
Before exposure, the oligochaetes were held in dechlorinated groundwater overnight to purge their guts of any previously ingested material prior to the initiation of the test. During the holding period, the sediment slurries were prepared by mixing dry (preweighed) Little Magaguadavic Lake (45°47.62'N, 67°13.48'W; near Fredericton, NB, Canada) sediment with dechlorinated groundwater that contained prepared aliquots of imidacloprid. Likewise, during the exposure period, sediment slurries devoid of imidacloprid were made ready for the subsequent transfer of oligochaetes from the treatment vessels (volume, 80 ml; as above). These postexposure egestion rates were used to estimate the reversibility of sublethal feeding effects. Lake Magaguadavic sediment was chosen because it had been used in previous L. variegatus studies and is known to be free of pesticides and other contaminants .
The L. variegatus egestion rate was measured by collecting surface-deposited fecal pellets generated by the consumption of sediment particles. Fecal pellets were deposited by oligochaetes on the sand layer separating the aqueous and sediment layers. Fecal pellets were then easily removed from the sediment surface with a glass transfer pipette at 1-d intervals. Four beakers containing sediment but devoid of worms were used to correct for pipetting error, whereby the overlying sand was transfer pipetted as though fecal pellets were being collected and the average value was subtracted from the daily measurements. Fecal material was filtered onto preweighed, 47-mm, glass-fiber filters (GF-C grade; Fisher Scientific), then dried at 110°C and reweighed to determine the dry mass of material egested per day (mg/oligochaete/d). Because the unit of measure is 1 d, the rate is expressed as μg/oligochaete throughout.
The present study examined imidacloprid at concentrations much less than the median detection limit of most commercial laboratories (∼2 μg/L). The various imidacloprid concentrations were created by diluting 1-ml aliquots of 240 g/L of Admire® (Bayer CropScience, Toronto, ON, Canada), then performing a standard serial dilution to achieve the desired treatment concentrations in dechlorinated groundwater. Chemical analyses of the imidacloprid samples were conducted at the National Water Research Institute (Environment Canada, Saskatoon, SK) on a Micromass Quattro Ultima liquid chromatography-mass spectrometer equipped with a stainless-steel column (MS Xterra C-8; 100 × 2.2 mm; Waters, Milford, MA, USA). Given imidacloprid's relatively high solubility (with respect to other pesticides; ∼510 mg/L), precautions were taken to account for matrix effects of the dechlorinated groundwater used in the creation of the treatment solutions by supplying the Saskatoon laboratory with monthly water-quality monitoring data for the New Brunswick water source. Samples for imidacloprid analyses were taken from three replicate exposure beakers (both mayflies and oligochaetes) per treatment concentration per experiment. These samples were collected in 40-ml, amber-glass vials (U.S. Environmental Protection Agency vials; Fisher Scientific) and stored at 4°C until shipment to the laboratory (within 10 d). The imidacloprid samples were injected directly into the liquid chromatography-mass spectrometry system. The mobile phase contained 40% aqueous acetonitrile and 0.2% formic acid (v/v). The flow rate was 200 μl/min, and injection volumes were 10 to 20 μl. Calibration of the instrument was performed on the stock solutions used for creating the test solutions. Results from the laboratory yielded a correlation between nominal and actual values of r2 = 0.999 for mayflies and oligochaetes, respectively. For may flies, the correlation of actual to nominal was y(actual) = 1.0019x(nominal) + 0.3098; for oligochaetes, the correlation of actual to nominal was y = 0.9979x — 0.2682. Actual values for both the mayfly and oligochaete experiments can be found in Table 1. Because of this high laboratory performance, concentrations are presented in nominal rather than actual concentrations throughout.
Table Table 1.. Comparison of the actual values (mean ± standard error [SE]) of imidacloprid as determined by liquid chromatography-mass spectrometry with respect to nominal
a No imidacloprid was detected in the 0.1 μg/L oligochaete treatment.
Standard toxicity tests were assessed using traditional LC50 and EC50 probit analysis (STATA Ver 8.02; SAS, Cary, NC, USA). Analysis of variance (ANOVA) was performed on feeding and egestion data using both the SAS (Ver 8.02) and Statistica (Ver 6; Statsoft, Tulsa, OK, USA) software packages. During the 24-h pulse (day-1) exposure, one-way ANOVAs were performed on the feeding and egestion rate data. The effect of imidacloprid treatment with respect to feeding and egestion rate over the 4-d recovery period was assessed repeatedly (days 2, 3, 4, and 5) from each replicate vessel using repeated-measures ANOVA. Repeated measures was chosen as an appropriate analysis because this model does not assume that the measurements were independent and could compare the rate changes to imidacloprid exposure, day, and number of surviving nymphs (or oligochaetes) per replicate treatment. Because imidacloprid induced mayfly mortality at the environmentally relevant concentrations that were previously thought to be sublethal, the feeding rate tests incorporated nymph number as a covariate in the statistical analyses. Assumptions of ANOVA, including normality (Shapiro-Wilk W test) and homogeneity of variance (Cochran's C test), were tested, and when these assumptions were not met, data were transformed and the residuals checked to ensure that test assumptions were met. Analyses were performed on both raw and transformed data. When significant effects were detected, the Bonferroni sequential post-hoc test was used [17,18]. The Bonferroni test incorporates the Bonferroni adjustment, which divides α by the number of tests conducted during post-hoc testing. All post-hoc testing was one-tailed and only evaluated whether the response variables are lower than the control because of treatment.
Mayfly test results indicated that imidacloprid is toxic to these larvae in the low-μg/L range. Early and late-instar mayflies had 24-h LC50s of 2.1 ± 0.8 and 2.1 ± 0.5 μg/L, respectively. The 96-h LC50 of late instars was 0.65 ± 0.15 μg/L. Oligochaetes were an order of magnitude less sensitive to imidacloprid, with a 96-h EC50 (immobility) of 6.2 ± 1.4 μg/L.
Mayfly feeding tests
Exposure to imidacloprid concentrations of 5 μg/L or greater caused significant reductions in early instar mayfly feeding during the 24-h pulse exposure (F5,23 = 4.70, p < 0.013) (Fig. 1A). Concentrations of imidacloprid as low as 0.5 μg/L also showed a trend toward reduction; however, these differences (0.5 and 1 μg/L) were not significant. No feeding was observed at concentrations of 10 μg/L because of mortality of all mayflies in the five replicate streams. Latent recovery to normal feeding levels could not be examined in these experiments because mortality (24-h LC50, 2.1 ± 0.8) and insufficient replication precluded comparing imidacloprid treatments over time. Treatments of 5 μg/L or greater had a significant decrease in nonconsumptive losses compared to the control level (F5,23 = 3.835, p < 0.05), suggesting a significant reduction in insect activity and feeding. In contrast, larvae in the 0.5 and 1 μg/L treatments exhibited higher nonconsumptive chlorophyll readings, which although nonsignificant may indicate less efficient scraping and feeding at these sublethal exposures (Fig. 1B). In replicate treatments that contained 10 μg/L of imidacloprid, only nonconsumptive losses were observed, likely because all the mayflies had died.
During the imidacloprid exposure, later-instar mayflies had reduced feeding rates in the 5 and 10 μg/L exposures, a trend consistent with the early instar results (Fig. 2). Even after correction for nonconsumptive losses, however, the later-instar mayflies appear to have increased feeding rates at some treatment levels (0.5 and 1 μg/L). This may suggest that increased feeding activity might be an adaptive response to low-dose imidacloprid exposure. This is consistent with the nonconsumptive losses observed in the early instar mayflies (Fig. 1B): Early instars were unable to incorporate the material, whereas later instars could. Whether the consumed Nitzschia sp. was suitably digested, however, was not evaluated in the present study.
After imidacloprid exposure in the day-1 treatment vessel, mayflies were transferred to flow-through artificial streams of the same dimensions as the test beaker. Postexposure, recovery feeding was significantly affected by imidacloprid treatment (F5,60 = 2.47, p = 0.042) and the number of days following exposure (F3,60 = 9.45; p < 0.001) (Fig. 3). Because all treatments (including control) had depressed feeding rates during the recovery (days 2–5) experiment as a result of handling, comparisons are only made within the 4-d recovery period (Fig. 3). Although transferring the mayflies in the laboratory between the exposure and test vessels did result in reduced feeding in the control group, the intense reduction in feeding rate during the recovery period demonstrates the feeding inhibition of mayflies exposed to imidacloprid.
Feeding rate of mayfly larvae was affected by time since imidacloprid pulse and exposure concentration (Fig. 3). Only larvae in the lowest-dose exposure (0.1 μg/L) recovered to control (day-5) feeding rates, although feeding depression was observed in this treatment on days 3 and 4. Mean feeding rates for larvae in treatments with an imidacloprid concentration equal to or greater than 0.5 μg/L decreased during the 4-d recovery period. Thus, significant sublethal effects were observed for up to 4 d after exposure and at an order of magnitude lower than what was observed following the initial 24-h imidacloprid challenge.
Oligochaete egestion tests
Oligochaetes were rendered immobile by 96-h (continuous) exposures to imidacloprid when concentrations exceeded 5 μg/L (96-h EC50, 6.2 ± 1.4 μg/L). Shorter (24-h) exposures of 10 μg/L or less, however, did not affect egestion or mobility (F5,42 = 149, p = 0.21) (Fig. 4). Following 24-h exposure to imidacloprid, animals in treatments with less than 1 μg/L increased egestion rate over the 4-d recovery period. As a result of handling, egestion rates initially were lower after each vessel transfer. Because oligochaetes were transferred twice, initially into exposure containers and later into recovery vessels, egestion was reduced at the beginning of the test. Treatments that had a sufficient dose of imidacloprid (>5 μg/L), however, maintained a depressed egestion rate for the duration of the test (F3,108 = 46.65, p < 0.001) (Fig. 5). The delayed recovery in the 0.5 μg/L (after day 2) and 1 μg/L (after day 4) treatments may suggest an extended period of anesthesia in annelids. Oligochaete recovery occurred at pulse concentrations an order of magnitude higher than those for mayflies (∼1 μg/L for oligochaetes vs ∼0.1 μg/L for mayflies).
Imidacloprid was designed to target invertebrate pests and is highly effective at selectively binding the insect nAcChR while being virtually nontoxic to higher vertebrates [8,9,19,20]. The nAcChR is common across insect taxa, however, because the origin of this receptor can be found deep in the evolution of neurochemical signaling . Therefore, this compound can affect both aquatic and terrestrial invertebrates. Mayflies and oligochaetes were chosen for the evaluation of this insecticide because they are important contributors to the functioning of lotic food webs [22,23] and can be exposed to imidacloprid either through rain-event pulses or contaminated groundwater. Imidacloprid exposure produced significant non-target effects in mayflies by causing mortality and by inhibiting feeding rate at low concentrations. In oligochaetes, imidacloprid induced immobility and reduced sediment egestion. Also, the effect of imidacloprid can be latent, with feeding and egestion inhibited upward of 4 d postexposure in some treatments.
This experiment demonstrated that mayflies exposed to 24-h pulses of imidacloprid greater than 0.5 μg/L did not recover to control feeding rates within 96 h (4 d) postexposure. Imidacloprid also caused mayfly mortality at environmentally relevant concentrations (24-h LC50, 2.1 μg/L) in both early and late-instar organisms. The use of the two mayfly life stages originally was conceived to account for potential tolerance differences in the different instar groups. Both groups had similar LC50s, however, which may reflect insignificant differences in the ability to metabolize imidacloprid between the two age classes. Because the upper concentrations in the recovery experiment incorporated the 24-h LC50, it was only with increased replication that sufficient later instars survived to compare the mayfly feeding inhibition over time. Therefore, the recovery results are the estimated feeding rate and recovery of less than 50% of the experimental E. longimanus population. These results are in keeping with population studies on the effects of imidacloprid, in which a 12-h pulse exposure of 10 μg/L or greater caused the loss of all male Epeorus from the adult cohort (A.C. Alexander, MSc thesis, University of New Brunswick, Fredericton, NB, Canada). Thus, the feeding rate at concentrations greater than the LC50 may reflect the continued feeding of the female mayflies only. Given the potential impact of imidacloprid on sex ratios and population viability, further study is warranted.
Because mayflies only feed as larvae, reduced consumption of foodstuffs can result in hampered larval development, reduced emergence, and smaller adult imagoes [24–26]. Nonconsumptive losses result from grazing disturbance , and our nonconsumptive results suggest that mayflies were less active in moderate to high imidacloprid exposures (5 and 10 μg/L) and, alternatively, exhibited increased activity in response to low imidacloprid exposure (0.5 and 1 μg/L). Feeding was reduced for at least 4 d following exposure to insecticide concentrations as low as 0.5 μg/L. This may be caused by the tremor and/or anesthetic action of imidacloprid , which would interfere with foraging activity. We hypothesize that low doses of imidacloprid (0.5 and 1 μg/L) induce mild tremors that increase nonconsumptive losses and reduce foraging efficiency, whereas higher concentrations produce more severe tremors, which limit foraging activity. Ultimately, larval mayflies were unable to recover to preexposure feeding rates (∼ 160 μg/mayfly ) during the 4-d recovery period, and the observed response could be either immediate (>5 μg/L) or latent (>0.5 μg/L). Feeding rates also decreased because of handling between the exposure and recovery periods. Mayflies in low-dose exposures, however, resumed feeding, similar to the control organisms, whereas mayflies in higher-dose exposures did not. Altered activity patterns are important, because other species may be affected, for example, changes in grazer-dislodged material (nonconsumptive losses) may be an important source of energy for downstream consumers [27–29].
Oligochaetes were more tolerant than mayflies of imidacloprid (oligochaete 96-h EC50 was 10-fold higher than the mayfly 96-h EC50) and were not affected during the 24-h pulse exposure in the egestion experiment. The 4-d postexposure experiment validated the 96-h EC50 (immobility) value of ∼6 μg/L (aqueous exposure), with no recovery to normal egestion rates (∼12 mg/worm ) observed for concentrations of 5 [μg/L or greater. Oligochaete egestion rate was reduced at the onset of the tests because of handling. As above, however, oligochaetes in low-dose exposures recovered to baseline egestion rates similar to the control organisms, whereas oligochaetes in higher-dose exposures did not. The response of oligochaetes to imidacloprid 96 h following exposure was primarily that of prolonged reduced egestion, lethargy, and immobility, whereas mayflies exhibited tremors, paralysis, and death . This taxonomic difference in response may be caused, in part, by functional differences in the acetylcholine receptors of insects versus annelids.
Previous research identified imidacloprid as an insecticide of concern in agricultural watersheds . Studies seeking to measure imidacloprid in surface waters, however, have encountered difficulty acquiring water samples that contain the compound. Because of the relative solubility of imidacloprid, it may pass through river systems at concentrations greater than analytical detection thresholds primarily during storm discharge events. Likewise, imidacloprid is difficult to detect with the chromatographic columns currently in use for the detection of more common nonpolar pesticides. Even when the detection of imidacloprid is possible using modified techniques, the detection limit employed in many commercial laboratories (∼2 μg/L) is well above the concentrations at which we detected effects on aquatic biota (∼0.5 μg/L). Clearly, improved monitoring and detection limits for imidacloprid are needed to track concentrations in the field. A similar problem arises with the detection of imidacloprid metabolites. Imidacloprid degrades into several nonpolar and highly persistent derivatives from either photodegradation [30,31] or decomposition (>400 d) [6,31]. To our knowledge, these derivatives are not currently being examined by regulatory agencies. Considering the toxic potential of these metabolites, future research should examine whether these metabolites accumulate in lotic food webs.
The present study was intended to evaluate the occurrence of sublethal responses caused by environmentally relevant concentrations of imidacloprid. Subsequently, we have determined not only that are reductions in feeding rate occurring but also that adult abundance and body size is reduced when larval mayflies are exposed to low-dose, pulse exposures of imidacloprid . Presently, these endpoints can be explained best by sublethal effects of imidacloprid exposure on feeding. Combined with the larval mortality associated with imidacloprid exposure, it seems likely that this insecticide could have impacts on the abundance and success of mayflies in streams. Consequences of reduced mayfly activity potentially include differential grazing in streams , whereas consequences of reduced mayfly abundance, especially of dominant taxa such as heptageniid mayflies , could include reduced foodstuff availability for fish.
Because delayed responses appear to be common for oligochaetes with respect to imidacloprid treatment, an even longer-term experiment may be warranted. This may be particularly salient, because soluble imidacloprid is a potential groundwater contaminant. Therefore, long-term, low-dose imidacloprid exposure may be common in sediment-dwelling organisms. Also, laboratory studies have identified a number of nonpolar metabolites of imidacloprid that may contribute to depressed egestion rates because of residual toxicity [30,31]. Given the sublethal effectiveness of imidacloprid, further studies may yield indications regarding the impact of this insecticide on oligochaete success in streams. For example, the reduction in oligochaete movement could increase predation risk by limiting the ability to avoid capture , whereas a reduction in egestion rates has the potential to alter organic matter processing and bioturbation in streams .
Imidacloprid exposure reduced the survivorship, feeding, and egestion of mayflies and oligochaetes at concentrations from 0.5 to 10 μg/L. Concentrations of imidacloprid greater than 5 μg/L limited oligochaete movement (24-h EC50, 6.2 ± 1.4 μg/L) and reduced egestion rate, with no recovery occurring in 4-d postexposure (>5 μg/L) experiments. Imidacloprid exposures at or exceeding 0.5 μg/L over a 24-h period caused significant long-term reductions in the feeding rate of late-instar mayflies that did not recover to control levels even 4 d after exposure. Of concern is that all these effects, including the mayfly 24-h LC50 of approximately 2 μg/L, were measured at exposure concentrations that overlap with observed environmental concentrations (0.25 ± 0.07 to 15.88 ± 0.99 μg/L). In Atlantic Canada, imidacloprid is an important insecticide used in the protection of potato crops . Because Canada has adopted a seed-coat application technique for runoff protection of stream resources, spring rain events are thought to be the primary threat for large influxes of imidacloprid to streams in potato-farming regions. Because imidacloprid continues to be detected in agricultural stream surface waters (3.6% of samples; C. Murphy, 2006; Environment Canada, Charlottetown, PE), continued monitoring that employs lower analytical detection limits is warranted along with future examination of the persistence and toxicity of imidacloprid metabolites.
We would especially like to acknowledge the technical advice and assistance of J. Bailey, D. Halliwell, C. Casey, E. Irving, P. Williams, K. Heard, E. Luiker, and D. Hryn. Earlier drafts of the manuscript benefited from critical review by D. Baird, G. Benoy, K. Kidd, and three anonymous reviewers. Support for this research was provided by financial assistance to A.C. Alexander from the University of New Brunswick and the Science Horizons internship program; an Environment Canada Pesticide Science Fund grant to J. Culp, K. Liber, and A. Cessna; and a Natural Sciences and Engineering Research Council of Canada Discovery grant to J.M. Culp.