Toxicity of Antifreeze to Dreissena bugensis Veligers and Adults

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1 Toxicity of Antifreeze to Dreissena bugensis Veligers and Adults Karim Alame, Anna Boegehold and Donna Kashian Wayne State University Department of Biological Sciences Meg Thompson and Nick Phelps Minnesota Aquatic Invasive Species Research Center Objective: Evaluate the lethal toxicity of a propylene glycol based antifreeze (Freeze ban -50 Antifreeze) on adult Dreissena rostriformis bugensis (quagga mussels) and dreissenid veligers (Dreissena sp.) through a series of laboratory bioassays using freshly collected animals. Methods: Dreissenid adults and veligers were collected from the Detroit River on Belle Isle State Park, Detroit, MI ( N, W). This site was selected based on a study by Ram et al. (2011) where it was confirmed by both morphologic and by PCR analysis that the site consisted of >99% adult D. rostriformis bugensis, and adult dreissenid mussels were taxonomically confirmed to be D. rostriformis bugensis in As dreissenid veligers are planktonic and are not easily identified by species at this life stage, veligers collected at this site were identified down to the genus Dreissena. During each sample collection, in-situ water chemistry measurements of temperature, dissolved oxygen, conductivity, and ph were recorded using an YSI multi-meter. Adult mussels were scraped from a seawall at depths 1-3 m below the surface using a steel scraper at the same location where veliger samples were collected. After collection, adult mussels and veligers were transported back to the lab in coolers. Mussels were maintained in the laboratory using L glass aquariums filled with aerated and dechlorinated tap water at a density of 1 mussel per ml of water. Adult quagga mussels were fed every other day with Ankistrodesmus falactus. Veligers were collected by tossing a 63 μm mesh Wisconsin plankton net over the pier and allowed to be carried by the current before retrieval. All planktonic organisms were rinsed off the 63 µm net into a 1L Pyrex glass bottle using Detroit River water. This process was repeated five times to ensure adequate amount veligers. Detroit River water was also collected and taken back to the lab where it was filtered using a.22 µm filter and stored in a 5 C refrigerator to be later used for rinsing and dilutions. Plankton samples were rinsed twice with filtered river water through a 2 μm nylon mesh to isolate veligers from large particulates and then filtered through an 80 μm mesh to isolate the veligers from smaller particulates. Veligers at growth stages appropriate for toxicological studies typically measure between 80 μm and 2 μm (Ackerman, 1994). Veligers were then rinsed off the mesh 1

2 and collected in a glass beaker where they were allowed to acclimate for 24 hours before use in a 23 C environmental chamber using a 12:12 light and dark cycle. Acute veliger assay (2.5 hours) Veligers were placed into a 60x15 mm glass Petri dish and examined using an inverted microscope under 200x magnification. Cross-polarized light was used to illuminate plankton with calcareous shells and identify veligers from other plankton as a distinct black X easily identifies them. For the acute veliger assay, seven treatments were tested including the full concentration of FreezeBan -50 Antifreeze (0%) and dilutions of 50%, 25%, 20%, 15%, 12.5%, %, and a control of river water (0%) (n=12). Water collected from the Detroit River was used for the dilutions and control. Microplate wells were filled with 300 μl of the appropriate treatment. A live veliger was randomly chosen from the sample and carefully placed into each well using a μl Eppendorf micropipette. Each well was inspected for mortality every 30 minutes for 2.5 hours, each check taking 15 seconds. Veligers were marked as dead when veliger was not swimming and cilia were no longer beating. Removing the plate from the stage after every check minimized exposure to heat produced from the microscope light. Acute assays were replicated three times. Acute assays were used to determine a suitable concentration range to be used in chronic exposures. Chronic veliger assay (6 days) The 6-day chronic veliger assay followed similar methods as the acute assays, but with the addition of food. A green algae, Chlamydomonas reinhardtii, was included in dilutions for all treatments. The assays consisted of 6 treatments with dilutions of %, 7.5%, 5%, 2.5%, 1% antifreeze in a solution with filtered Detroit River water and a control consisting only of filtered Detroit River water (n=12). Each veliger was checked for mortality daily for 6 days by observing each well for 30 seconds. Due to evaporation, each well was replenished with new solution containing food on the third day. Chronic veliger assays were replicated three times. Acute Adult Assay (2.5 hours) For the acute adult assays, twelve adults (n=12) for each treatment (0%, 50%, 25%, 20%, 15%, 12.5%, % and 0%) were randomly placed in individual glass vials with 30mL of treatment, for a total of 96 vials. The vials were aerated through gentle stirring at every check in 15 minute intervals. Response to gentle stimuli was used as an indicator that a mussel was alive. Mussels were then determined to be dead if the mantle does not close when prodded with a sterile pipette tip. Acute assays were also used to determine a suitable concentration range for chronic exposures. Acute adult assays were replicated three times. Chronic Adult Assay (6 days) 2

3 Similar methods from the acute adult assay were used for the chronic 6-day mussel assay, but with the addition of food. Ankistrodesmus falactus was added to the dilutions for all treatments. This assay consisted of 5 dilutions of antifreeze and a control (%, 7.5%, 5%, 2.5%, 1% and 0%) with an n=12, and a total of 72 vials. Daily checks for mortality were performed by stirring each vial and carefully observing response to stimuli. Each mussel that did not respond to gentle stirring was checked by gentle prodding with a sterile pipette tip. On the third day, noticeable evaporation was observed and vials were replenished by carefully removing fluids and pipetting in new solutions. Data Analysis A log logistic analysis, described in Solla et al. (2017), was used to analyze mortality data for the chronic and acute assays on both veligers and adults to determine the lethal concentration at which 50% of the organisms died (LC 50 ). A Fisher s Exact test was used to see where there is a significant difference in mortality at each concentration compared to the control at the end of each assay. Results: Acute veliger assays: Veligers were highly sensitive to the antifreeze. In the acute assays all of the veligers died at concentrations of 0% and 50% within 30 minutes. Veligers in the 25% concentration had a high mortality within 30 minutes (86.11%) and all veligers died within 90 minutes. Veligers in the 20%, 15%, and 12.5% concentrations had increased mortality at 2.5 hours. There was no mortality in the % concentration or the control for the assay (Figure 1). The LC 50 of antifreeze for the acute veliger assays is 15.98% with an upper confidence of 19.27% and a lower confidence of 12.68% (Figure 2; p<0.01). Chronic veliger assays: Veligers in the chronic assay survived up to 24 hours in the % dilutions with zero mortality, but all veligers in % dilutions died by the end of day three. Veligers in the 7.5% dilutions, the second highest concentration, experienced 0% mortality by day 6 (Figure 3). While total mortality was not observed in the 5%, 2.5%, and 1% dilutions, veligers appeared impaired, showing little to no swimming after 48 hours of exposure. In contrast, veligers in the control maintained vigorous swimming throughout the course of the experiment. The LC 50 of antifreeze for the chronic veliger assays is 1.57% with an upper confidence of 2.80% and a lower confidence of 0.340% (Figure 4; p<0.05). Acute adults assays: 3

4 Adult mussels experienced immediate mortality when exposed to 0% concentrations of antifreeze during acute assays, while all mussels exposed to the 50% dilution expired within 45 minutes. Mussels exposed to the 25% dilution had a mortality of 80.56% at 2.5 hours. Mussels exposed to 20%, 15%, 12.5%, and % dilutions had increasing mortality over 2.5 hours (Figure 5). No deaths were observed in the control treatments. The LC 50 of antifreeze for the acute adult assays is 19.74% with an upper confidence of 25.34% and a lower confidence of 14.13% (Figure 6; p<0.01). Chronic adult assays: Similar to the veliger assays, adult mussels survived for 24 hours in the % and 7.5% dilutions. Likewise, all mussels in % dilutions died by the end of day three and all mussels in 7.5% dilutions died by day 6 (Figure 7). Mussels exposed to 5% dilutions had 80.56% mortality at day 6. Mussels exposed to 2.5% and 1% dilutions reached a mortality of 38.89% and 33.33%, respectively at the end of the assay. There were no deaths observed in the control treatments. Over the 6 days of exposure, across all concentrations of antifreeze, there was a noticeable reduction in response time to stimuli in mussels that remained alive. The LC 50 for the chronic adult assay was 1.87% antifreeze with an upper confidence of 3.24% and a lower confidence of 0.49% (Figure 8; p<0.05). 0 Mean Percent Mortality Time (minutes) Percent Anitfreeze 0.0% 50.0% 25.0% 20.0% 15.0% 12.5%.0% Control 4

5 Figure 1. Mean percent mortality of dreissenid veligers with standard error based on binomial means exposed to increasing concentrations of FreezeBan -50 Antifreeze during a 2.5 hour acute toxicity assay. Mortality was quantified every 30 minutes (n=12; p<0.01) based on a Fisher s Exact test between 0% and 20% Mean Percent Mortality % 50.0% 25.0% 20.0% 15.0% 12.5%.0% Control Concentration of Antifreeze LC 50 = 15.98% Figure 2. LC 50 of dreissenid veligers exposed to FreezeBan -50 Antifreeze during an acute 2.5 hour toxicity assay (n=12; p<0.01) based on a Fisher s Exact test between 0% and 20%. 5

6 Mean Percent Mortality Percent Anitfreeze.0% 7.5% 5.0% 2.5% 1.0% Control Time (Days) Figure 3. Mean percent mortality with standard error based on binomial means of dreissenid veligers exposed to increasing concentrations of FreezeBan -50 Antifreeze during a 6-day chronic toxicity assay. Mortality was quantified daily (n=12; p<0.05) based on a Fisher s Exact test between 0% and 2.5%. 6

7 Mean Percent Mortality % 7.5% 5.0% 2.5% 1.0% Control Concentration of Antifreeze LC 50 = 1.57% Figure 4. LC 50 of dreissenid veligers exposed to FreezeBan -50 Antifreeze during a chronic 6-day toxicity assay (n=12; p<0.05) based on a Fisher s Exact test between 0% and 2.5%. Mean Percent Mortality Percent Anitfreeze 0.0% 50.0% 25.0% 20.0% 15.0% 12.5%.0% Control Time (minutes) Figure 5. Mean percent mortality of adult dreissenid mussels with standard error based on binomial means exposed to increasing concentrations of FreezeBan -50 Antifreeze during a 2.5 hour acute toxicity assay. Mortality was quantified every 15 minutes (n=12; p<0.01) based on a Fisher s Exact test between 0% and 25%. 7

8 Mean Percent Mortality % 50.0% 25.0% 20.0% 15.0% 12.5%.0% Control Concentration of Antifreeze LC 50 = 19.74% Figure 6. LC 50 of adult dreissenid mussels exposed to FreezeBan -50 Antifreeze during an acute 2.5 hour toxicity assay (n=12; p<0.01) based on a Fisher s Exact test 0% and 25%. 8

9 Mean Percent Mortality Percent Anitfreeze.0% 7.5% 5.0% 2.5% 1.0% Control Time (Days) Figure 7. Mean percent mortality of adult dreissenid mussels with standard error based on binomial means exposed to increasing concentrations of FreezeBan -50 Antifreeze during a 6- day chronic toxicity assay. Mortality was quantified daily (n=12; p<0.05) based on a Fisher s Exact test between 0% and 2.5%. 9

10 Mean Percent Mortality % 7.5% 5.0% 2.5% 1.0% Control Concentration of Antifreeze LC 50 = 1.87% Figure 8. LC 50 of adult dreissenid mussels exposed to FreezeBan -50 Antifreeze during a chronic 6-day toxicity assay (n=12; p<0.05) based on a Fisher s Exact test 0% and 2.5%. Conclusions: This assay was successful in examining the toxicity of a propylene glycol based antifreeze across both veliger and adult life stages of dreissenid mussels. Mortality was high in both adults and veligers at very low concentrations. In the adults we determined the chronic LC 50 to be 1.87% (p<0.05). The veligers were more sensitive to the antifreeze and we determined the chronic LC 50 to be 1.57% (p<0.05). Antifreeze is highly toxic to Dreissena bugensis and is successful in causing high mortality at low concentration.

11 Literature Cited: Ackerman, J.D. (1994). A review of the early life history of zebra mussels: comparisons with marine bivalves. Canadian journal of zoology. 72 (7):1169. Gilroy, È. A.M., Gillis, P. L., King, L. E., Bendo, N. A., Salerno, J., Giacomin, M. and de Solla, S. R. (2017), The effects of pharmaceuticals on a unionid mussel (Lampsilis siliquoidea): An examination of acute and chronic endpoints of toxicity across life stages. Environ Toxicol Chem, 36: Ram, JL, Karim, A.S., Acharya, P., Jagtap, P., Purohit, S., and Kashian, D.R. (2011) Reproduction and genetic detection of veligers in changing Dreissena populations in the Great Lakes. Ecosphere 2, art.3 11