Abstract:
We examined lethality and behavioral effects of Corexit EC 9500A (C-9500A) exposure on the model marine zooplankton Brachionus plicatilis singularly and in combination with abiotic and biotic factors. C-9500A exposure at standard husbandry conditions (17.5ppt, 24°C, 200 rotifer*mL-1 density) identified the 24 hr median lethal concentration, by Probit analysis, to be 107 ppm for cultured B. plicatilis. Rotifers surviving exposure to higher concentrations (100 and 150 ppm) exhibited a decreased swimming velocity and a reduced net to gross movement ratio. Significant interaction between C-9500A exposure and temperature or salinity was observed. Upper thermal range was reduced and maximal salinity stress was seen as ca. 25ppt. Increased or decreased nutritional availability over the exposure period did not significantly alter mortality of B. plicatilis populations at concentrations tested. Results from this study may be useful for predicting possible outcomes on marine zooplankton following dispersant application under diverse natural conditions.
Data Parameters and Units:
One spreadsheet with 7 tabs. LC50: concentration (ppm), observations, number Temperature: concentration (ppm), temperature (degrees C), number of survivors (out of 100) Salinity: concentration (ppm), salinity (ppt), number of survivors (out of 100) Population Density: concentration (ppm), density (rotifers per ml), surviving (out of 100) Feeding Rationing: concentration (ppm), ration (ratio of normal feed ration), surviving (out of 100) Swimming Speed: concentration (ppm), speed (um*sec-1), observations, time point (h) Swimming Gross the Net: concentration (ppm), net/gross movement, time point (h)
Methods:
Maintenance of Stock Populations A stock population of the rotifer species Brachionus plicatilis was established from populations obtained originally from Reed Mariculture Inc. (Campbell, CA). Rotifer stock was maintained under constant aeration and a 12 hr light, 12 hr dark photoperiod at a 10L volume, 17.5ppt salinity, and 25°C. Rotifer stock was provided an AM (09:00) and PM (17:00) feeding of a concentrated Nannochloropsis alga paste (Reed Mariculture Inc. Campbell, CA) at a concentration of 0.3 mL*L-1 and 0.6 mL*L-1 respectively. To maintain water quality baseline (<0.1 mg/L Cl, <3 mg/L P, and <1 mg/L NO2−) and exponential growth in the population, prior to each AM feeding 20% of the volume with its associated rotifers was removed and replaced with 2L of 17.5ppt artificial sea water (ASW) (Instant Ocean, United Pet Group, Blacksburg, VA). Determination of LC 50 A 1L volume of B. plicatilis was removed from stock prior to AM feeding and average population density was calculated from samples collected between 10:00 and 11:00 hr (n=5 1mL samples were counted and averaged). Density counts do not account for any population growth between the time when samples are collected and when the rotifers are exposed. The separated 1L of rotifers was provided 0.3 mL/L concentrated Nannochloropsis and maintained at 24°C until time of exposure. Exposures were always initiated at 15:30 in the afternoon. ASW (17.5ppt) was added to increase volume to reach a final population density of approx. 200 ind.*mL-1. This population represents various life stages including juveniles and adults. B. plicatilis were exposed in 20mL glass vials at concentrations of C-9500A ranging from 0 to 300ppm (following initial range finding bioassays) (n=5 vials / treatment concentration) produced by serial dilution and all vials capped with plastic paraffin film. B. plicatilis populations were exposed for a 24 hr period at 12 hr light, 12 hr dark photoperiod in a 24°C incubator. Over the 24 hr exposure period vials received an AM and PM feeding of 0.3 mL*L-1 and 0.5 mL*L-1, respectively, and an O2 gas application (bubbled 5 secs directly into each vial using a glass pipet). Following the exposure period vials were shaken and rinsed with ASW and contents emptied onto a glass Petri dish. The contents were again mixed with a glass pipet to homogenize the contents and 1mL was removed to assess % alive and % dead. The % alive and % dead was determined for each replicate by observation of the first 100 rotifers to be located in the field of view under light microscope (Nikon SMZ1000, Nikon Inc. Melville, NY). Alive was determined by motility through the water or movement of the mastax or foot, any of which are common indicators of viability (ASTM, 1998). The same experiment was replicated in 3 separate trials and outcomes among trials were averaged for further analysis. Behavioral Response In this experiment we used the same exposure methodologies used to determine medial lethal concentration with some modifications. B. plicatilis populations were exposed in a 22°C incubator to 0, 50, or 100ppm concentrations of C-9500A for 6, 12, or 24 hrs (n=5 vials / treatment concentration at each exposure period). Following exposure periods the vials were removed from the incubator and moved to a 23-24°C ambient temperature and lighting. Five 1mL aliquots from each treatment vial were placed into 3mL of clean ASW of matching temperature and salinity on glass petri dishes (radius of 23mm). A field of view observing 1-12 rotifers was recorded for 5-7 secs at 45 frames per second (fsp) at a resolution of 600x800 pixels by light microscope using a Moticam 2000 microscope camera and Motic Images Plus 2.0 software (Motic North America, British Columbia Canada). Video was analyzed in CellTrak 1.5 motion analysis software (Motion Analysis Co. Santa Rosa, CA) for average swimming velocity (µm*sec-1) and average net to gross movement (distance from starting location divided by total distance traveled, where a value of 1 would represent perfectly straight line of travel) over the time period. C-9500A Lethality at Suboptimal Temperature In this experiment we used the same exposure and mortality methodologies used to determine medial lethal concentration with some modifications. B. plicatilis populations were exposed to 0, 50, 100, or 150ppm concentrations of C-9500A (n=2 vials / treatment concentration). Exposures were completed in either low (11 - 26°C) or high (24 - 40°C) temperature range maintained by an aluminum block (61x23x8cm) that is heated from one side and cooled from the other to create a linear temperature gradient extending an ca. 15°C range. The same experiment was replicated in 2 trials and outcomes among trials were averaged for further analysis. C-9500A Lethality at Suboptimal Salinity In this experiment we used the same exposure and mortality observation methodologies used to determine median lethal concentration with some modifications. Prior to the experiment the B. plicatilis stock was segregated into 4 separate stocks and acclimated over a 21 day period to salinities of 5, 17.5, 25, or 32ppt by adding ASW of appropriate salinity (separate stocks maintain equal population growth during acclimation). Once acclimated and raised to a 10L volume they were maintained as described previously. B. plicatilis populations at each of these 4 salinities were exposed in a 22°C incubator (Percival) to 0, 50, or 100ppm concentrations of C-9500A (n=3 vials / treatment concentration). The same experiment was replicated in 2 trials and outcomes among trials were averaged for further analysis. C-9500A Lethality at Suboptimal Population Density In this experiment we used the same exposure and mortality observation methodologies used to determine medial lethal concentration with some modifications. ASW (17.5ppt) was added to increase water volume to reach approximate population densities of 50, 200, and 350 ind.*mL-1 at the initial time of exposure. B. plicatilis populations at each of these 3 population densities were exposed in a 22°C incubator to 0, 50, or 100ppm concentrations of C-9500A (n=5 vials / treatment concentration). The same experiment was replicated in 2 trials and outcomes were averaged for analysis. C-9500A Lethality at Sub-Satiating Feeding Regime In this experiment we used the same exposure and mortality observation methodologies used to determine medial lethal concentration with some modifications. B. plicatilis populations were exposed in a 22°C incubator to 0, 50, or 100ppm concentrations of C-9500A (n=3 vials / treatment). Over the 24 hr exposure period vials received an AM feeding of 0, 0.15, 0.3, 0.45, or 0.6 mL*L-1 and PM feeding of 0, 0.25, 0.5, 0.75, or 1mL*L-1 respectively. The same experiment was replicated in 2 trials and outcomes among trials were averaged for further analysis. Statistics 24 hr LC50 was calculated by Probit Analysis. Velocity and net to gross movement were compared by a weighted multi-factorial ANOVA to test for interaction between levels of C-9500A exposure and duration of exposure. Post-hoc of velocity and net to gross movement between groups within the same time period were compared by a weighted one-way ANOVA. Bonferroni correction was applied to compensate for repeated comparisons reducing the level of significance to p < 0.00263 for all velocity and net to gross movement statistics (Dunn, 1961). All other comparisons were performed by multi-factorial ANOVA (p < 0.05 significance) on aligned rank transformed data as described by Wobbrock et al. (2011) to test for interaction between abiotic or biotic secondary stressors and C-9500A exposure. All analyses were computed using IBM SPSS statistical software package ver. 20.
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