The effects of Earth Tec, a molluscicide, on zebra mussel (Dreissena polymorpha) mortality

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1 The effects of Earth Tec, a molluscicide, on zebra mussel (Dreissena polymorpha) mortality Madeline Genco 1 and David Wong 2 ABSRACT Zebra mussels are a major biofouling pest in the United States. In this study the effect of Earth Tec, a copper based molluscicide, was evaluated for mortality on both adult and larval mussels. Various concentrations of the chemical were tested on adults and veligers for differing durations of time in order to determine a necessary dosage for use in water treatment plants to prevent clogging of intake pipes as mussels colonize. Results have shown 100% mortality of adult mussels exposed to 8ppm of Earth Tec for 96 hours and at 16ppm for 72 hours. However, such a trend was not seen with the veligers, which were less sensitive to treatment. INTRODUCTION Zebra mussels (Dreissena polymorpha) are a major biofouling pest in Otsego Lake and much of the United States. They were accidentally introduced into the Great Lakes via ballast water in 1986, and spread from there (Herbert et al. 1989). They were first discovered in Otsego Lake in 2007 (Horvath 2008). They cause many ecological problems in lakes and streams in the United States. They are outcompeting native mussels for food and space (Karatayev et al. 1997). They clear the water, altering the depth at which light reaches, which in turn increases plant growth (Hecky et el. 2004). Adult mussel attachment to pipes becomes a problem and can lead to clogging (Wong et al. 2012; LePage 1993). The Otsego Water Treatment Plant has been having problems with their intake pipe clogging due to the overabundance of zebra mussels attaching within them (Coyle et al. 2015). Earth Tec, a commercially available biocide, could be a possible solution to this problem. It uses a blend of copper sulfate pentahydrate with a base acid. The cupric ion (copper 2+) is the active ingratiate; this is a more biologically available form of copper (Watters et al. 2013). Copper 2+ also does not precipitate out of solution, meaning that more copper remains in the water and a lower concentration may be used as compared to copper sulfate or other copper based chemicals. Earth Tec was recently approved for use to combat invasive dressenid mussels and was selected as Top 10 Water Technology at the American Water Works Association (AWWA) Conference (Earth Tec website). The purpose of this experiment was to determine if the chemical Earth Tec can be used in low concentration to kill adult and veliger zebra mussels and be a viable alternative to chlorine or potassium permanganate to prevent clogging of water treatment intake pipes. 1 Biological Field Station Intern, Summer Current affiliation: SUNY Oneonta 2 Assistant Professor and Biological Field Station Researcher, SUNY Oneonta Biology Department.

2 METHODS Adult mussels The methods described here were developed concurrent with a study by Coyle et al. (2015). Mussel collection Before mussels were collected, 20 small (20 L) tanks and two large holding tanks were cleaned with an abrasive sponge, rinsed, and refilled with lake water. The tanks were allowed to soak with the lake water overnight and then dumped out and left empty until mussel collection commenced. The two large holding tanks were set up with a constant flow of lake water to keep them at the ambient temperature of the lake. Twenty four hours prior to mussel habitation, the 20 small tanks were refilled with lake water and set up with air stones calibrated for equal air flow between all tanks. Each of the 20 small tanks were labeled with the tank number, and the concentration in parts per million of Earth Tec to be placed within it. Adult zebra mussels were collected from the Thayer Farm Boat House on Otsego Lake, a site having suitable (rocky) substrate. Rocks were removed from about 2 meter deep water via snorkeling. Adult mussels were removed by hand and placed into a bucket until approximately 2,000 mussels were collected. The mussels were mixed in order to prevent bias; however, mussels connected via byssal threads were left intact to reduce stress on the mussels. Mussels were rinsed with lake water before being placed in mesh drawstring bags (Comeau et al. 2011; Watters et al. 2013; Coyle et al. 2015). Preparing the mussels Eleven mussels were placed in each mesh bag, making 160 bags total. The bags were strung across dowels with 8 bags on each dowel. The dowels held the bags together within the tank (dowels laid across the top of the tanks allowing the bags to drape down into the tank (see Figure 1)). Eighty bags were then placed in each of the holding tanks and allowed to acclimate to conditions for 3 days. Before beginning the experiment, the bags were checked for dead mussels. Any dead mussels were removed. If no mussels were dead, the smallest mussel was removed. If more than one mussel was dead, the dead mussels were replaced with live ones from a bag of extra mussels. After removing dead or extra mussels, each bag was left with 10 mussels. Each bag was labeled with the tank number, the concentration of Earth Tec in the tank, and the number of hours after which it was to be removed from the tank and transferred to the holding tank. Conducting the experiment The day of the experiment, Earth Tec at concentrations of 0,1,4,8, and 16 ppm was added to the 20 small tanks (see Table 1). Each concentration/duration was evaluated in quadruplicate. A dowel with 8 bags strung across it was then added to each of the 20 small tanks (see Figure 1). One bag of mussels from each of the 20 small tanks was removed, rinsed with

3 lake water, and placed into the large holding tanks at each time interval listed in Table 2. Bags were removed from tanks beginning with tanks having the lowest concentration of Earth Tec up to the highest concentration. Forty eight hours after being moved to the holding tanks, mussels were removed from the tanks and dried with paper towels. Mussels were then checked for mortality. A mussel was determined dead if it did not immediately shut its shell after being gently pulled open. Shell lengths were measured in millimeters using a hand caliper. A spreadsheet was used to record which mussels were dead and alive, as well as to record the shell lengths. Figure 1. Experimental tank set up for adult mussels. Each of the 20 small tanks has a dowel set over the top with 8 mesh bags of mussels attached and draping down into the water. An air stone was placed in the corner of each tank (after Coyle et al. 2015). Table 1. Adult mussels: Concentration of Earth Tec in each tank is listed. Tank # [Earth Tec ] Tank # [Earth Tec ] 1 0 ppm 11 0 ppm 2 1 ppm 12 1 ppm 3 4 ppm 13 4 ppm 4 8 ppm 14 8 ppm 5 16 ppm ppm 6 0 ppm 16 0 ppm 7 1 ppm 17 1 ppm 8 4 ppm 18 4 ppm 9 8 ppm 19 8 ppm ppm ppm

4 Table 2. Adult mussels: Schedule for the time interval at which different parts of the experiment were performed. The first two columns show the times at which bags were removed from the tanks with Earth Tec into holding tanks and YSI readings were taken. The last column shows when the same bags were checked for mortality and measured 48 hours later. Time Passed Mussels moved from small tanks to holding tanks and YSI readings measured: Mussels removed from holding tanks and checked for mortality and length measured (note: this is 48 hours after they were removed from 20 small tanks with chemicals). 0 hrs. Tues. 6/24/14 12pm Thurs. 6/26/14 12pm 3 hrs. Tues. 6/24/14 3pm Thurs. 6/26/14 3pm 6 hrs. Tues. 6/24/14 6pm Thurs. 6/26/14 6pm 12 hrs. Wed. 6/25/14 12am Fri. 6/27/14 12am 24 hrs. (1 day) Wed. 6/25/14 12pm Fri. 6/27/14 12pm 48 hrs. (2 days) Thurs. 6/26/14 12 pm Sat. 6/28/14 12pm 72 hrs. (3 days) Fri. 6/27/14 12 pm Sun. 6/29/14 12pm 96 hrs. (4 days) Sat. 6/28/14 12pm Mon. 6/30/14 12pm Veligers The day of the experiment, twenty eight 250mL beakers were set up in a grid as shown in Figure 2. Each beaker was filled with 200mL of room temperature lake water that had been filtered with a 63 um net. Zebra mussel veligers were collected from Otsego Lake using a 63 um plankton net. Veligers were collected from Otsego Lake (about halfway across the lake from The SUNY Oneonta Biological Field Station Main Lab dock) with 8 vertical pulls of the net from a depth of 13 meters. A flow meter on the net was used to determine how much water was filtered. The sample was poured sieve to strain out any large daphnia. The sample was concentrated using a cup with a 63 um mesh bottom (Watters et al. 2013; Coyle et al. 2015). This cup was lowered into a beaker containing the sample, and water was withdrawn using a 10mL pipet with the end of the tip cut off to prevent exposing the veligers to strong suction. One ml of the condensed sample was viewed under the microscope. Cross polarized light microscopy and a gridded Sedgewick Rafter slide was used to make the veligers easier to detect (Johnson 1995). The number of veligers on the slide was counted and that number was used to estimate how many ml of sample was needed in each beaker so that each contained about 200 veligers. This part of the experiment required 2 people: Differing amounts of Earth Tec was added to each beaker corresponding with the concentrations shown on the grid in Figure 2. No

5 chemicals were added to beakers in the 0 minute column; these beakers were controls. Addition of chemicals to each row of beakers was staggered by 5 minutes. This was to account for the amount of time it took to look at the contents of each beaker under the microscope. About 3 minutes before a sample was to be viewed under the microscope, it was concentrated in the beaker using the same methods as before. After the sample in a given beaker was condensed, 1 ml of the remaining fluid was pipetted onto gridded Sedgewick Rafter slide. The samples were viewed on the slide under polarized light at 5 minute intervals beginning with the bottom left most beaker on the grid and moving up until all beakers in the column had been viewed under the scope. The next column would begin at the time listed on the grid. During the 5 minutes each slide was under the microscope, the number of dead and living veligers was counted. If no movement of organs or internal fluid was seen within 4 seconds, the veliger was considered dead. Figure 2. Experimental set up for veligers. Each circle represents a 250 ml beaker having the specified treatment concentrations and durations of Earth Tec (after Coyle et al. 2015).

6 RESULTS Adult mussels An analysis of variance (ANOVA) was conducted to determine the effect of time and concentration of Earth Tec on adult mussel mortality. It was found that both time exposure and concentration of Earth Tec significantly affected mortality (Figure 3). No statistical difference was found between mortality at 1 ppm and 0 ppm; however, statistical differences were seen between all other concentrations. No statistical significance was found between mortality at 0, 3, and 6 hours, nor between 48 and 72 hours. However, statistical significance was found between all other time points. It was also found that there was no difference in size between alive and dead adult mussels (T-test, α=0.05, P = 0.88). This means that mussel size is not correlated with mortality, ruling out mussel size as a possible confounding variable. Figure 3. Comparison of different concentrations of Earth Tec and how these concentrations affect the mortality rate of adult mussels given the amount of time they were exposed to the chemical. Veligers The ANOVA conducted to determine the effect of time and concentration of Earth Tec on veliger mortality does not show any statistical difference between the chemical treatments (F = 1.40, P = ). However the effect of time is statistically significant (F = 4.18, P = ) (Figure 4). This means that while the treatment dose had no effect on mortality, the amount of time passed did. There is no difference between times 0-24 hours. However, there is a difference between 48 hours and 0-24 hours. There is also a difference between 144 hours and 0-48 hours (Figure 5).

7 Figure 4. A comparison of different concentrations of Earth Tec and how these concentrations affect the mortality rate of veligers given the amount of time they were exposed to the chemical. Figure 5. A comparison of concentrations of 0 and 1ppm of Earth Tec and how these concentrations affect the mortality rate of veligers given the amount of time they were exposed to the chemical. A long term point (144 hours) was used here.

8 DISCUSSION Earth Tec is effective at killing adult zebra mussels in relatively low concentrations. However, concentrations needed to induce mortality in veligers were higher than anticipated. The veliger results are interesting because 8 ppm and 6 ppm are considered fairly high doses of Earth Tec, yet the mortality rate was still not as high as what has been reported elsewhere. A previous study by Watters et al. (2013) showed 100% mortality in quagga mussel (Dreissena rostriformis bugensis) veligers after 30 minutes at 3ppm. Due to this previous finding, we anticipated our zebra mussel veligers would be susceptible to much lower concentrations and shorter durations than what was seen here. A possible explanation for the low mortality rate is that there may be highly variable developmental stages of veligers in the samples that were used during the experiment. Some stages of veligers may be more susceptible to the copper than others. As veligers develop, they secrete shell material; the older veligers have thicker shells (Choi et al. 2014) (Figure 6). Later stage veligers are also larger in size. It could be that later stage veligers would be less susceptible to copper. The sample used for experimentation has variable veliger stages (Table 3). Preserved samples for trials 2 to 4 were measured, and the 4 th trial had the lowest proportion of pediveligers (Table 3). ANOVA shows the 4 th trial had the highest mortality rate than other two trials (F = 49.2, P < 0.001) although no difference was found among treatment (F = 2.96, P = 0.061). This could explain the low mortality rate, compared to a sample being of mostly first stage veligers. While higher mortality may be associated with younger veligers, various stage veligers found in the lake are the stages that will be brought into the water treatment plant pipe. Therefore, chemicals used in the water treatment pipe must be able to kill all veliger stages. Figure 6. Veliger stages from left to right: trochophore, straight-hinged veliger, umbonal veliger, and pediveliger.

9 Table 3. Veliger stages were observed and recorded from preserved sample. Samples were preserved using ethanol. Veliger stages were determined using the descriptions from Choi et al Veligers Trial 2 (7/21/14) Trial 3 (7/24/2014) Trial 4 (8/1/14) # of veligers % of each stage # of veligers % of each stage # of veligers % of each stage Stage 1 Trochophore Stage 2 D-Stage Stage 3 Umbonal Stage 4 Pediveliger REFERENCES Choi, W.J., S. Gerstenberger, R.F. McMahon, and W.H.Wong Estimating survival rates of quagga mussel (Dreissena rostriformis bugensis) veliger larvae under summer and autumn temperature regimes in residual water of trailered watercraft at Lake Mead, USA. Management of Biological Invasions 4(1): Claudi, R., T.H. Prescott, S. Mastisky, and H. Coffey Efficacy of copper based algaecides for control of quagga and zebra mussels. RNT Consulting, Inc. Comeau S, S. Rainville B., Baldwin, E. Austin, S.L. Gerstenberger, C. Cross and W.H. Wong Susceptibility of quagga mussels (Dreissena rostriformis bugensis Andrusov) to hot-water sprays as a means of watercraft decontamination. Biofouling 27: Coyle, B.P., P.H. Lord, W.H. Wong and M.F. Albright Preventing zebra mussel colonization: appropriate potassium permanganate application time and dose. In 47 th Ann. Rept. (2014). SUNY Oneonta Biol. Fld. Sta., SUNY Oneonta. Earth Tec QZ website. Hebert P.D.N., B.W. Muncaster and G.L. Mackie Ecological and genetic studies on Dreissena polymorpha (Pallas): A new mollusc in the Great Lakes. Canadian Journal of Fisheries and Aquatic Sciences. 46: Hecky, R.E., R.E.H. Smith, D.R. Barton, S.J. Guildford, W.D. Taylor, M.N. Charlton, and T. Howell The nearshore phosphorus shunt: a consequence of ecosystem engineering by dreissenids in the Laurentian Great Lakes. Can J Fish Aquat Sci 61:

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