The mosquito larvicide methoprene is a juvenile growth hormone mimic that is widely used to control mosquito larvae in stormwater catch basins. This study addresses two concerns pertaining to methoprene's use for mosquito control. First, measurements of methoprene concentrations were made from water in catch basins that had been treated with methoprene and from an adjoining salt pond near where the treated catch basins emptied. The concentrations of methoprene in catch basins and at drainage outlets after application at the rates currently used for mosquito control in southern Rhode Island were 0.5 ppb and lower, orders of magnitude below what has been determined as detrimental to organisms other than mosquitoes. Second, the effects of methoprene on the communities that live in catch basins were evaluated both in simulated catch basins in the laboratory and in actual catch basins in the field. We found no evidence of declines in abundances of any taxa attributable to the application. Furthermore, we found no consistent changes in community-level parameters (e.g., taxonomic richness, and dominance-diversity relationships) related to methoprene application in either field or laboratory trials.
The Rhode Island Department of Environmental Management has used methoprene formulated as Altosid® 30-day slow-release pellets with 4.25% active ingredient (Wellmark International, Dallas, TX) applied at a rate of 3.5 g per catch basin per month for mosquito control. When the flushing of pellets from catch basins following a simulated rainfall event was studied, no pellets were found captured on mesh secured over the end of an outflow pipe after meticulous examination of the captured debris (A. Gettman, unpublished data). This suggested that the roughly 24 pellets (3.5 g) remained in the catch basin where they were administered, but the dissolved chemical was not addressed.
The first purpose of this study was to measure the concentration of methoprene found in catch basins and at outflow sites when 3.5 g of pellets were applied for mosquito control. Methoprene can be flushed into natural waters when the briquet or pellet is washed out, when the dissolved chemical is rinsed out, or the product might be bound to other particles that are transported out. In this study we address the second mode of transport by measuring methoprene concentrations in treated catch basins and, after simulating a rain fall, from an outflow area in Point Judith Pond, Narragansett, RI. Secondly, we measured the effects of methoprene on communities of organisms that live in catch basins both in the laboratory and in the field.
MATERIALS AND METHODS
Methoprene field concentrations
Levels of methoprene found in catch basins and at outflow sites were measured one week after methoprene was applied for mosquito control. During the summer of 2001, two connected catch basins that empty into Point Judith Pond on Harbor Island, Narragansett, RI were each treated with 3.5 g of pellets, the concentration used by the Rhode Island Department of Environmental Management for mosquito control. This amount is based on surface area rather than volume of water as per product label instruction. Both catch basins were 123 cm by 123 cm, but the depth of the water found in each differed. Catch basin 1, the shallower catch basin (about 35 cm deep) flowed into a canal that flowed directly into Point Judith Pond, and catch basin 2 (about 61 cm deep) flowed under a street into catch basin 1. Water samples used for calibration of the gas chromatograph (GC) and the Method Detection Limit (MDL) study were collected from Point Judith Pond about 30 m from the outflow site from a small dock before methoprene sampling began. The standard protocol for chemical analysis of trace compounds such as methoprene requires that an MDL be calculated. The MDL is defined by the American Industrial Hygiene Association (AIHA) as the minimum concentration of an analyte that, in a given matrix and with a specific method, has a 99% probability of being identified, qualitatively or quantitatively measured, and reported to be greater than zero (Botnick and White 2002). MDLs for methoprene were calculated by two different laboratories that independently analyzed our samples, Southern Testing Laboratories (STL) (Wilson, NC), contracted by Wellmark International and CEIMIC (Narragansett, RI), an analytical chemical laboratory located near the study site.
One week after catch basins were treated, when methoprene concentrations were expected to be at their highest (Ross et al. 1994), water samples were collected for methoprene analysis from both catch basins prior to the flooding event. Next, using a fire hose, approximately 5,000 liters of water were rinsed into the upstream catch basin (2), flushing the contents of both catch basins into Point Judith Pond. Immediately following the simulated storm event, replicate water samples were taken from Point Judith Pond at the outflow site and approximately 30 m away from the outflow area from a dock. After one h the two sampling sites in the pond were re-sampled. All samples were taken from just below the surface in an effort to obtain maximum levels of methoprene (Schaefer and Dupras 1973).
Methods for methoprene extraction and concentration determination (gas chromatography) were modified from the EPA Method #616 (EPA). Seventy-five ml of methylene chloride were used for extraction and samples were kept on ice while being transported to the two independent laboratories for analysis. One set of samples (two replicates from each sample site) was analyzed by STL, and two sets of samples (four replicates) were analyzed by CEIMIC. Method Detection Limits were established at 0.20 ppb (STL) and 0.18 ppb (CEIMIC); therefore only measurements above these values were valid within the 99% CI.
Community effect field study
Thirty catch basins from two sites in Narragansett, RI were sampled during July by taking a biological sample and measuring environmental variables including temperature, pH, conductivity, dissolved oxygen, depth, and the amount of carbon, nitrogen, and total suspended solids per liter. Biological samples were taken using a custom-made catch basin sampler that collected a sample of the water column from the surface to the benthos (see Butler et al. 2007 for details). The catch basins were found mostly on street corners that were roughly 100 m apart from each other. Each street corner had between one and four catch basins. After sampling, half of the catch basins were treated with 3.5 g of pellets each. Catch basins were specifically chosen for treatment in such a way that water from treated catch basins did not flow into control catch basins. At one site (Rodman Street), 16 catch basins were connected along a single road. The ten upstream catch basins were not treated and the six downstream catch basins were treated. At the second site (Mettatuxett Road), 14 catch basins were located on three parallel streets where one street (five catch basins) was not treated and the nine other catch basins were treated. One month later and again two months later, catch basins were sampled to determine if methoprene had an effect on the catch basin communities. Samples taken one month later (August) were compared to samples from July (prior to treatment) since it was during these months that organisms were most abundant. Biological samples were brought back to the laboratory where soft-bodied organisms were fixed in near-boiling water for approximately one min and then the entire sample was preserved in 70% ethanol. After 24 h, the ethanol was replaced with fresh ethanol. Organisms were later sorted from debris, enumerated, and identified to lowest possible taxonomic level. Because annelids were often broken, their abundance was estimated as biomass. Abundances for all other taxonomic groups were calculated as number per liter, and communities were characterized by counting the number of different types of organisms identified to lowest taxonomic category. Since we were not always able to identify organisms to species, we refer to this as “taxonomic richness” rather than “species richness.” Dominance-diversity curves were plotted to graphically show abundances of dominant organisms as well as evenness (Southwood 1966).
Two-factorial ANOVAs were used to measure the effects of time (before and after), treatment (control and treated with methoprene), and the interaction of time and treatment on taxonomic richness and on individual taxonomic groups for samples collected in July (n = 25) and in August (n = 26). Significant differences in time alone and treatment alone would not show an effect of the addition of methoprene to these communities. However, a significance of the interaction term in this “Before After Control Impact” (BACI) experimental design, where samples are taken before and after the disturbance (addition of methoprene) and under treated and control conditions (Bernstein and Zalinsky 1983, Stewart-Oaten et al. 1986), would indicate a significant effect of the addition of methoprene. Because of frequent violations of assumptions, we performed the ANOVA on transformed data (log(X+1)). We also assessed possible changes in the proportion of catch basins in which a taxon was present using G-tests (Sokal and Rohlf 1969).
Community effect laboratory study
Artificial catch basins were constructed in the catch-basin simulation laboratory (CBSL) at the University of Rhode Island. The basement laboratory housed ten cement catch basin replicates, each roughly one-fourth the size of an actual catch basin (approximately 48 cm wide × 82.5 cm long × 57 cm deep). Two approximately six-week-long experiments were conducted, one beginning on June 13, 2003 and the second beginning on August 14, 2003. Experimental organisms were collected from several catch basins in Narragansett that had not been treated with methoprene for at least two years using a dip net and a bucket. Ten insulated coolers were filled with debris (including benthic organisms), organisms from the water column, and water and transported back to the CBSL. In the laboratory, the contents of each cooler were thoroughly mixed and divided evenly into each of the ten catch basin simulators (CBS). Roughly 20 cm of concentrated debris was deposited in each CBS and then each was filled to approximately 72 liters (30 cm deep) using municipal, non-chlorinated water. Next, half of the CBSs were selected randomly and treated with 0.6 g of methoprene formulated as Altosid® 30-day slow release pellets and half were left as controls. This amount was determined to be proportional to the 3.5 g of pellets used in the field to treat catch basins for mosquito control. The experiments were conducted at ambient light, temperature, and humidity. Temperatures ranged from 15 to 22° C during the first experiment and from 19 to 23° C during the second experiment. Each catch basin was covered with a screen, and as the amount of water in the catch basins decreased, water was not replenished.
One week after methoprene application, water samples were taken for methoprene analysis. The first set of approximately 1-liter (954 ml) samples were fixed with 75 ml of methylene chloride and then frozen for transport to Wellmark International's laboratory. The samples from the second experiment were not fixed, but rather were put on ice and immediately sent to Wellmark International for analysis. Sampling in a manner similar to that described in the community field study was conducted weekly, and after approximately six weeks, CBSs were sampled a final time and then entire contents were drained through a 164 μm sieve. Organisms were preserved in 70% ethanol. A two-factorial-ANOVA (BACI experimental design) was used to analyze the laboratory data and significance of the interaction (time × treatment) term indicated an effect of the methoprene application. Again, data were transformed by log(X+1) to stabilize the variances. Organism abundances and taxonomic richness on the day the experiment started were compared to organism abundances and taxonomic richness six weeks later when the experiment was terminated.
Methoprene field concentrations
Of the 36 field water samples analyzed by the two laboratories, only one of the measurements taken from one catch basin had a level of methoprene greater than the MDL. STL found 0.50 ppb methoprene in the water of one of the catch basins. All other values reported were below detection (Table 1). STL also measured methoprene acid, a breakdown product of methoprene in the samples, but all values were below their MDL for methoprene acid (0.50 ppb). CEIMIC analyzed four replicates from each sampling site without detecting any methoprene at levels above their MDL (0.18 ppb). However, trace amounts of methoprene (0.05 to 0.12 ppb) may have possibly been detected in samples taken from both catch basins and in two of the four samples taken at the outflow immediately following the simulated rainstorm given the values observed and the experimental design.
Table 1. Chemical analysis of water samples for methoprene content by CEIMIC labs and Southern Testing Labs (C-CEIMIC labs, S-Southern Testing Labs).
Community effect field study
Dominance diversity plots for control and treated catch basin communities for July (prior to treatment) and August (one month after treatment) are shown in Figure 1. In July before treatment, graphs from treated and control catch basins show mosquito larvae dominating the community followed by copepods. Bivalves were only found in the treated catch basins and Isopoda, Collembola, and Oribatida were found in similar abundances in both treated and control catch basins. In August, one month after treatment, high abundances of mosquito larvae were found in several treated catch basins (mean ± standard error; 26.61 ± 2.87 per liter) compared to the control catch basins (5.43 ± 1.15 per liter). One of the treated catch basins had 123 mosquito larvae per liter and five treated catch basins had more than ten mosquitoes per liter. In the control catch basins, the most mosquitoes found in a single catch basin was 48 per liter with only two having greater than ten. In August, aside from mosquito larvae and copepods, other taxonomic groups were represented similarly in both treated and control catch basins.
The three-way interaction of month × treatment × taxa was not significant (F= 0.183; df = 12; P= 0.999), but we could not stabilize the variances, even with transformed data (Levene's test; F= 10.306; df1 = 51; df2 = 611; P < 0.001). Therefore, we performed two-way ANOVAs (month × treatment) with log(X+1) transformed data on changes in community level parameters and abundances of individual taxa. There were no significant interaction terms (time × treatment) for either taxonomic richness (both when mosquito larvae were included and omitted) or for abundances of individual organisms (Table 2). Therefore, no statistically significant effect of the methoprene on the taxonomic richness or abundance was detected by this study.
Table 2. Results of 2-factorial ANOVA for field study. Significance of interaction term (month × treatment) indicates effects from methoprene addition.
Aside from the interaction term, we saw that overall, more taxonomic groups were found in treated (5.9 taxa) catch basins than in untreated (4.8 taxa) catch basins (F= 4.049; df = 1; P≤ 0.05) both before and after treatment. However, individually, this difference occurred only at the Mettatuxett site (ANOVA; F= 4.101; df = 1; P= 0.052). At Rodman Street, where the 16 catch basins were connected, there was no significant difference in taxonomic richness between treated and control catch basins (ANOVA; F= 1.822, df = 1 P= 0.185). Furthermore, the position of the catch basin along the chain of connected catch basins did not appear to influence taxonomic richness (Figure 2).
One taxon, Hydracarina, was more abundant in August samples than in July samples in both treated and untreated catch basins (F= 13.176; df = 1; P= .001). The proportions of catch basins in which various taxa were present were not affected by methoprene treatment (Table 3), except for Collembola, which increased in frequency in the treated catch basins.
Table 3. Effect of methoprene treatment on presence/absence of major taxa in catch basins studied in the field.
Proportion of Catch Basins with Taxon Present
G= value of G for 3-way interaction between treatment/control × before/after treatment × number of catchbasins with taxon present/absent (df = 1).
Community effect laboratory study
In both laboratory experiments, the dominance diversity relationships showed shallow curves at the beginning of the experiments with organisms relatively evenly distributed among taxonomic groups. Six weeks later, both treated and non-treated communities were dominated by copepods (Figures 3 and 4). Taxonomic richness and abundances of several taxa changed (F > 31.424, df = 1; P < 0.001) from the start to the end of the experiment. However, there was no significant effect of methoprene treatment, because the interaction term was never significant in this study (Table 4).
Table 4. Results of 2-factorial ANOVA for two laboratory studies. Significance of interaction term (month × treatment) indicates effects from methoprene addition.
Our results suggest that methoprene applied to catch basins in 30-day slow release pellets is not flushed into natural waters in detectable amounts (≥ 0.2 ppb). Furthermore, our results suggest that the use of methoprene is not likely to affect non-target organisms in natural environments when used according to label instructions. Since methoprene concentrations were below detectable levels in Point Judith Pond, our study area, it is extremely unlikely that it would be found in larger bodies of water such as Long Island Sound or Narragansett Bay at levels anywhere near detection. Our data show that the only organisms likely to be exposed to methoprene at detectable levels (≥ 0.2 ppb) are those that share catch basin environments with mosquito larvae.
Detecting methoprene in the field has proven a challenge in many studies since minimal detectable levels are so close to the levels used for mosquito control (Sjogren et al. 1986, Knuth 1989, Woodrow et al. 1995). For example, although Hershey et al. (1995) used a formulation different from ours (150-day slow release briquets) in their freshwater pond study, an overall average of 0.5 ppb was found in their samples, with high variability ranging from none detected to greater than 45 ppb in similarly treated and sampled ponds.
Another possible reason for the high variability in methoprene concentration is the amount of organic matter in our replicates, given methoprene's affinity for organic matter (Kamei et al. 1992). In our first laboratory community experiment, no detectable methoprene was measured from samples sent to Wellmark International, and in the second experiment again all five water samples from treated simulated catch basins measured below detection. However, when organic particles were analyzed for methoprene content, measurable amounts were recorded ranging from 5.7 ppb to 15.4 ppb (See Table 1). Measuring methoprene content in water samples is not, in itself, a complete quantification of methoprene in the environment. Methoprene bound to organic matter needs to be considered in addition to methoprene in the water to properly assess the availability of methoprene to mosquito larvae and other organisms, especially since particulates can settle, potentially increasing concentrations in bottom sediments (Walker et al. 2005).
Research on the effects of methoprene on various organisms has shown effects at concentrations of less than 2.0 ppb primarily on mosquitoes. One exception is Mysidopsis bahia, the opossum shrimp, which showed reduced numbers of young produced per female at levels as low as 2.0 ppb (McKenney and Celestial 1996). In another study, Walker et al. (2005) showed that while mortality rates were essentially the same as that for the Stage II lobster larvae controls kept at 0.1 and 0.5 ppb (approximately 14%), mortality rate for larvae kept at 1 ppb increased to 30% and at 10 ppb mortality was 86%. Whether these apparent effects were statistically significant is not clear, yet these are among the lowest concentrations shown to possibly be deleterious to non-target organisms. Nevertheless, they are still far greater than the concentrations we measured in water samples taken at outflow areas immediately after catch basins were thoroughly rinsed into Point Judith Pond in our study (<.18 ppb). Decomposition and dilution undoubtedly reduce methoprene levels even further (Schaefer and Dupras 1973, Quistad et al. 1975, Schooley et al. 1975, Boxmeyer et al. 1997). The non-target organisms in natural waters did not appear to be at risk from methoprene pellets applied to catch basins according to what we found in this research. More research into other, more sensitive methods for obtaining reliable estimates of methoprene concentrations in water and sediment samples would help confirm these results.
Overall, our analyses of invertebrate communities and of abundances of various taxa detected no negative effects of methoprene treatment on the invertebrate faunas of catch basins. Considering the relatively high amounts of methoprene needed to elicit responses from most invertebrates other than mosquito larvae, it was not surprising that we were unable to measure a negative effect of methoprene on aquatic communities in either field or laboratory experiments. Furthermore, this study was not designed to evaluate methoprene's effects on mosquito larvae since methoprene does not remove mosquito larvae from the aquatic community. Treated mosquitoes molt to larger larvae or die when they are unable to emerge from the pupal stage to the adult stage (when they would be leaving the aquatic community anyway). This study may have, however, counted some organisms that were killed by methoprene and then preserved in the sample. We assume that organisms we captured were living when they were captured, but it is possible that some organisms were already dead either from methoprene or other causes. Furthermore, organisms killed by methoprene would remain in the immature stage. In this study we looked at both adults (e.g., oribatida, collembola, isopods, copepods) and larvae and saw no differences in either density.
Taxonomic richness was lower in control than in treated catch basins, possibly due to our selection of catch basins that were treated and those left as controls. We opted not to randomly choose catch basins to treat, because if methoprene were added to upstream catch basins, rain might carry the pesticide downstream contaminating control catch basins. Therefore, we treated catch basins in such a way that treated catch basins did not flow into control catch basins. At the Mettatuxett site where three separate parallel chains were found, two of the chains (nine catch basins) were treated with methoprene and a single chain (five catch basins) was left as a control. Significantly lower taxonomic richness was observed from these five control catch basins (4.6) compared to the nine other treated catch basins (6.0). This finding suggests that perhaps it was the locale rather than the treatment that accounted for this lower taxonomic richness in the control catch basins. This could be due to any number of factors not considered in this study, for example, other pesticides or lawn chemicals used by home owners, steepness of slope, or amount of tree cover.
The lack of difference in taxonomic richness at the Rodman Street site supports this explanation. In addition, since the catch basins at the Rodman Street site are connected, if there had been a significant increase in taxonomic richness in control catch basins at the bottom of the chain of catch basins, location on the chain and the lack of independence between catch basins might have been a concern. However, the lack of significance and a plot of taxonomic richness vs position in the catch basin chain showing no downstream trend in species abundance (Figure 2) suggest that organisms are not accumulating downstream.
These findings suggest that although the catch basins are connected, and perhaps not completely independent of each other, there is no evidence that their lack of independence is causing taxonomic richness to differ based on location of catch basins in the chain. These data together with the supporting evidence from the laboratory study, where catch basins were not connected and therefore were independent, suggest that the addition of methoprene had no significant effect on these field communities in this study. The only place where a difference in taxonomic richness was noted appears to be due to the locale of the five control catch basins at the Mettatuxett site.
The statistical analyses from the laboratory studies (Table 4) verify what is clearly shown in Figures 3 and 4. The addition of methoprene did not affect these communities in this study. The diversity of the community dramatically declines over time with copepods dominating the community by the end of the experiment. This may be an artifact of the experiment because there was no opportunity for recruitment of other organisms. We do not know why copepods predominated in both of these experiments, and this population explosion of copepods may have masked any impact that methoprene might have had, but nonetheless, we did not measure any difference between what occurred in the treated catch basin simulations compared to the control catch basin simulations.
Our research also supports the extensive multi-year project examining effects of methoprene on freshwater pond communities in Minnesota at least for their first year of sampling (Hershey et al. 1995, 1998, Hanowski et al. 1997a, 1997b). They found no detrimental effects of methoprene on birds that used the ponds for feeding and breeding or on zooplankton populations (Hanowski et al. 1997a, 1997b, Niemi et al. 1999). Also, methoprene had no significant effects on insect densities and biomass the year treatment started. However, in the subsequent two years, some insect densities were significantly lower partly due to a decrease in the Diptera, the insects targeted by methoprene, but some predator numbers declined in some cases, too. Species richness also declined in the second and third years after treatment (Hershey et al. 1995, Hershey et al. 1998). This study suggests that it may take longer than a season or even a year to see effects of methoprene on communities. Temporary communities that are frequently repopulated from external sources such as those living in urban drains (Kikuchi 1992) or salt marshes (Lawler et al. 2000) may be less susceptible to methoprene than more permanent communities such as the ponds studied by Hershey et al. (1995, 1998). If it takes two to three years to see effects of methoprene on a community and the majority of the organisms in catch basins are there on a much shorter time scale, effects of methoprene are likely negligible when compared to other catastrophic occurrences, such as a rain storm. Perhaps the relatively short-lived communities that exist in catch basins are not exposed to methoprene long enough for substantial effects.
We are grateful to the following faculty, staff, and students at the University of Rhode Island: Maria Aliberti, Katie Allen, Dr. Steven Alm, Dr. David Bengtson, Adam Butler, Dr. Richard Casagrande, Melanie Cheeseman, Charles Dawson, Caryn Debatt, Linda Green, Mary McDougle, Dr. Neeta Pardanani, Fred Pollnak, Carl Sawyer, Jesse Siligato, Cannsotha Suom, and Jane Viera. We also thank Nimish Vyas and Paula Henry and three anonymous reviewers. This work was funded by the Rhode Island Agriculture Experiment Station Hatch Grant #RI00666, URI's Coastal Research Fellowship Program, and the Northeastern Mosquito Control Association John L. McColgan Grant In Aid. Use of trade or product names does not imply endorsement by the U.S. Government.