1) 30 µg/L Atrazine: The peak concentration of atrazine detected in any sample within the Chesapeake Bay was 30 µg/L (station L0002488), which is located in a tributary on the Eastern Shore shown in Supplementary Figure 1 . (US EPA 2006). The highest concentration recorded is of interest, as although rarely reported due to lack of available testing, it is still found within the watershed.
2) 20 µg/L Atrazine: A half-way point in between 30 µg/L Atrazine and 10 µg/L Atrazine for data exploration purposes.
3)10 µg/L Atrazine: During June of 2011, atrazine concentrations from the upper Chop Tank River were logged. The average of all recorded concentrations was 0.29 µg/L. The highest recorded concentration was 10 µg/L (Chesapeake Bay Program; USDA: Hively et al. 2011). The general trend in the data indicates that the main detections of atrazine are in the tributaries while significantly lower concentrations have been found in the Bay itself. Oyster restoration grounds are generally found within tributaries along the upper, middle and lower sections of the Choptank river (Spires 2015; Scharping et.al 2016; Clement 2016, Christopher G., et al. 2016).
4) 3µg/L Atrazine: The EPA’s Maximum Residue Limit (MRL) for atrazine is relevant because it is the limit which the EPA has set forth for regulating our own drinking water, further investigation into the MRL of atrazine is merited based on health concerns cited in many scholarly papers throughout the last decade (Hussein et.al 1996; Trentacoste et. al 2001; Pogrmic-Majkic et.al 2018; U. S. EPA MRL).
5) 30µg/L Acetone: Atrazine stock solution was dissolved in a 100% acetone solution. In order to assume continuity throughout the experiment this treatment was added to assure that any noticed effect on development was due solely to atrazine exposure.
Oyster Acquisition and Stabilization
One specimen acquisition and stabilization event took place for this study in the Fall of 2018. In this experiemnt, 250 - 300 juvenile Crassostrea virginica oysters of similar size and weight were purchased from Horn-Point Laboratory. The oysters were separated into five groups of 50 specimens and randomly assigned to an exposure group (30 µg/L Atrazine, 10 µg/L Atrazine, 3 µg/L Atrazine, 30 µg/L Acetone and a Control). For the second event, a sixth exposure group was added: 20 µg/L Atrazine. 3.0 mm square mesh sieves were used to separate each group. No oyster was smaller than 5.0 mm long x 4.0 mm wide when placed in the mesh sieves. For each stabilization event, a large holding tank was filled with 300L of pressure filtered water and raised to a salinity of 25 parts per thousand (ppt). For the stabilization event, oysters were allowed to grow in the lab within this stabilization tank for a period of two weeks. Frequent water changes (25% twice weekly) were used in order to minimize buildup of both ammonium and nitrate within the closed water system . In addition to frequent water changes, Kordon AmQuel Plus Ammonia Detoxifier/ Conditioner and TLC Saltwater aquarium conditioner were used in order to remove Nitrate, Nitrite and Ammonia as needed. Each oyster group was fed 6L of a concentrated phytoplankton mixture of (Tetraselmis Chuii, isochrysis galbana, and Nannochloropsis oculata) approximately ~ 400,000 cells/mL) every every day.
Relevant tank water parameters were monitored and adjusted as needed by replacing old 25ppt saltwater with new 25ppt saltwater. The tank was consistently maintained to fit the water quality parameters outlined in Table 1 below:
Tabel 1. Salinity (ppt) Ammonium (ppb) Nitrate (ppb) Plankton Concentration 25 0 0 > 9,000 cells/ml |
Water quality parameters were monitored using:
(1) ]Salinity Refractometer - Salinity
(2) API Testing Kit - Ammonium levels
(3) API Testing Kit - Nitrate levels
(4) Mass Spectrophotometer at 654nm - Plankton Concentration
(5) Hemocytometer count - Plankton Concentration