Antipyrine mussels

Uptake and Elimination. Six mussels were placed into 600 ml beakers containing 300 ml aerated Instant 0ceanR (10-12°C) for a 1/2 hr acclimation period before addition of antipyrine (3 pCi/ beaker) (or other test compound). Aliquots (1.0 ml) were taken at intervals and placed directly into scintillation fluid (10 ml, 3a70B, Research Products International Corp., Elk Grove Village,... 

IL). Initial experiments showed that uptake was complete within 2 hours. At the end of 2 hours (uptake) the mussels were rinsed, antipyrine solution was replaced with fresh Instant 0ceanR, and sampling at intervals continued. Portions of the water were also analyzed for antipyrine and metabolites. Data expressed in counts per minute per 1.0 ml were used to represent uptake and elimination of antipyrine. Experiments were routinely done in triplicate. 

Graphs relating antipyrine concentrations and time were used to calculate clearance rates. A relationship between apparent antipyrine steady state concentrations at 120 and 240 minutes (api 2 o, ap2 o) and mussel body water and mantle cavity water was also determined (k). Mantle cavity water is that volume held between the valves when the mussels are closed, e.g., when transferred from the uptake solution (300 ml) to the elimination solution (300 ml). The initial antipyrine concentration (apo) was determined at the beginning of the experiment. Assuming no loss of antipyrine, complete mixing of the solutions, and its distribution into total mussel body water, when an apparent steady state is achieved, the following results ... 

Similarly, at achievement of the second apparent steady state following transfer of the antipyrine treated mussel to 300 ml water, the following equation can be written ... 

Quantitative estimation of ventilation by indirect methods in mussels requires four assumptions (16) a) reduction of concentration results from uptake, b) constant ventilation (pumping) rate, c) uptake of a constant percentage of concentration (first order process), d) homogeneity of the test solution at all times. Our transport studies have utilized antipy-rine (22, 23) a water soluble, stable chemical of low acute toxicity to mussels. It is readily dissolved in ocean water or Instant Ocean and is neither adsorbed nor volatilized from the 300 ml test system. Mussels pump throughout the 4 hour test period and this action is apparently sufficient to insure homogeneity of the solution. Inspection of early uptake and elimination curves (antipyrine concentration as a function of time) prompted use of Coughlan s equation (16) for water transport. 

Antipyrine from 300 ml solutions containing 0.6, 6, or 60pM was readily taken up by mussels. Within 40-80 minutes an apparent steady state was achieved. Uptake experiments were routinely conducted for 120 minutes (Figure 1). An analogous antipyrine elimination curve is shown in the lower portion of the figure. 

Figure 1. Antipyrine uptake and elimination data taken from 3 experiments. Six mussels in 300 mL Instant Ocean were used in each. The 1 mL aliquots were taken at the end of each interval.

Figure 2. Semilog plot of antipyrine uptake data of Figure 1. Each set of points is from a separate experiment using 6 mussels in 300 mL Instant Ocean ...

Apparent Steady State Antipyrine Concentrations and Calculated Mussel Mantle Cavity and Body Water... 

Days in Laboratory apo Antipyrine - (cpm/ml) ap120 ap240 2 Mussel Water (ml) V If 120 240 ... 

Mussels held at 11°C in aerated Instant Ocean. Antipyrine (6 ViM 1.65 mCi/mmole) in 300 ml. One ml aliquots taken at zero, 120 (uptake), and 240 (elimination) min. Three beakers per set. 

Three sets of experiments have been done to determine the effect of environmental variables on antipyrine disposition. The conditions included 1) animals transferred to either Instant Ocean or ocean gater antipyrine solutions, 2) animals maintained in Instant Ocean in the laboratory for periods up to 31 days, 3) mussels placed into antipyrine solutions which contained low levels of other foreign compounds (aldrin, p-nitroanisole, SKF 525-A). Results are summarized in Tables IV and V. 

Antipyrine uptake and elimination half-Jives were measured in both Pacific Ocean water and Instant Ocean. Measurements were made immediately after collection of the mussels (0 day). 

The uptake and elimination half-lives of 176 and 169 min and 27 and 29 min were similar to each other and to half-lives obtained using mussels maintained in the laboratory. Half-lives in the longer term laboratory culture experiments (Table IV) were similar to each other. Similarly, the mantle cavity and body water constants gave no indication of stress (Table II). Mussels used in these experiments were selected by size (ca. 6 g viscera fresh weight) and variability could be reduced by adoption of more objective criteria. Instant Ocean culture does not directly effect antipyrine disposition and laboratory conditions are suitable for maintenance of animals for at least short times. 

Antipyrine uptake and elimination was also assessed in the presence of aldrin and p-nitroanisole, among substrates used in biotransformation studies (Table V). Altered antipyrine half-lives might indicate that the levels of the substrates were stressful to the mussels. The levels used did not overtly effect the mussels pumping activity, and half-lives were within the range of control values. Antipyrine disposition measurements will be included in studies on the biological fate of chemicals. 

Antipyrine Uptake and Elimination by Mussels Directly Removed or Laboratory Cultured. 

Effect of Aldrin, p-Nitroanisole, and SKF 525A on Uptake and Elimination of Antipyrine by Mussels... 

Estimated from linear portion of uptake and elimination curves (semilog). Six mussels per 300 ml containing 1.8 ymole antipyrine (0.6 pM). 

These studies with aldrin and antipyrine are sufficient to document the in vivo oxidative metabolic capability of mussels. Limits of activity have not been established, but they will be explored by further varying dose and the duration of the test period. With these limits established, the influence of environmental stressors such as salinity, and dissolved and/or suspended particulate matter in water on biotransformation will be assessed. If biotransformation processes are affected by these conditions, their measurement may provide results which can be diagnostically used as indicators of environmental quality. 

---