Physiological and molecular effects of individual and mixtures of drugs of abuse in Crassostrea virginica larvae.

Drugs of abuse (DAs) are increasingly detected in surface waters, yet their effects on marine organisms remain poorly understood. This study evaluated the impacts of environmentally relevant concentrations (100 and 1000 ng L⁻¹) of cannabinol, codeine, and methamphetamine, individually and in mixtures, on the growth and development of Crassostrea virginica larvae. Eight-day exposures significantly reduced larval survival, impaired growth, and altered swimming behavior, with mixtures producing the strongest effects. At 1000 ng L⁻¹ , cumulative survival reached zero in mixture and cannabinol groups, while methamphetamine and codeine reduced survival by 74.8% and 56.0%, respectively. Drug mixtures also caused the greatest reduction in larval growth (13.3%) and mean swimming velocity (67.5%) relative to controls, with significant shifts from predominantly rectilinear swimming to circular or motionless behavior. Short-term (24 h) exposures induced changes in the expression of stress-related genes (mapk14, hsp70, sod1, and gst), with hsp70 exhibiting the strongest response (14- to 39-fold upregulation), indicating rapid activation of oxidative stress and detoxification pathways, particularly under mixture conditions. These findings demonstrate that DAs, especially in combination, can disrupt physiological and molecular processes in oyster larvae at concentrations reported in surface waters. Given their persistence, increasing consumption, and documented occurrence near wastewater discharges, DAs warrant consideration as contaminants of emerging concern in coastal ecosystems and should be incorporated into ecological risk assessments that account for mixture effects and early-life stage sensitivity.