Despite the lack of behavioral changes following exposure to NSAID pharmaceuticals, crayfish had significant physiological impacts. Female crayfish exposed to ibuprofen and naproxen had higher amplitude and time constant values for their oxygen consumption models than non-exposed females indicating an altered physiology. Amplitude in this context corresponds to the amount of oxygen being consumed at any given point whereas the time constant corresponds to the rate at which oxygen was consumed. In the lowest (0.30 µg/L) and highest (30 µg/L) concentrations of both ibuprofen and naproxen treatments, females consumed more oxygen and at a faster rate than the control females. These results indicate that assessing the biological impacts of some toxicants should be done at multiple levels of biological organization because at one level (behavioral), organisms may not show effects that are present at a secondary level (physiology).
This study utilized measurements at two different biological organizational levels to assess impairment from pharmaceutical exposure. By including both behavioral and physiological assays, there is an increase in the potential to detect more subtle effects of exposure at sublethal concentrations, especially in non-target organisms. A singular assay, particularly at higher levels of biological organization, may not be sensitive enough to determine the toxicity of a contaminant, especially when delivered in concentrations that are below established NOEC or LOEC standards. While behavioral assays may be easier to perform, as well as EC50 or LC50 assays, smaller physiological effects could remain undetected, producing effect concentrations that may be incorrect. Additionally, because animal behavior can change as a result of physiological effects, including a physiological measurement of impairment may aid in predicting what types of behavioral impairments can follow contaminant exposure.
While changes in oxygen use and consumption may seem like subtle toxicological effects, these small changes can add up to larger population and ecosystem effects. Chronic exposure to contaminants that change oxygen usage or food consumption could eventually disrupt a crayfish’s ability to maintain homeostasis as well as perform more energy intensive behaviors important to their ecology, such as burrow construction and aggression. Crayfish that consume more oxygen to create shelters and defend themselves are more likely to exhaust energy supplies. If so, other physiological processes related to crayfish reproduction can also be negatively impacted by changes to oxygen use and food consumption. Exoskeleton molting and regeneration are both tasks that require a significant amount of energy and are crucial for growth and mating. If animals are hindered in their oxygen consumption, these critical functions may become lethal. By modulating oxygen use, crayfish may not be able to successfully perform a suite of behaviors essential for sustaining their populations. Shifting the population dynamics of crayfish has implications to aquatic communities. In freshwater habitats, crayfish occupy a substantial ecological niche, as they can alter macrophyte abundance (Chambers et al., 1990), aid in nutrient cycling through processing of detritus (Parkyn et al., 2001; Usio & Townsend 2004, Creed & Reed 2004), and redistribute sediment along stream banks (Creed & Reed 2004). While we are unclear of the toxic mechanism by which NSAIDs act to change oxygen use in crayfish, these impacts add to the growing body of literature concerning effects of pharmaceuticals on non-target organisms.
As a broad class of chemicals, pharmaceuticals have an even broader range of physiological effects than just oxygen consumption (Arnold et al., 2014). Given that there is still growing evidence of unintended consequences on target organisms (primarily humans), there should be unexpected and unpredictable effects on non-target organisms (Arnold et al., 2014; Boxall et al., 2012). One example of unpredictable effects on non-target organisms related to pharmaceutical exposure is the major decline of the Gyps vulture populations on the Indian subcontinent (Swan et al., 2006). Veterinary use of diclofenac (another common NSAID compound) in livestock led to vultures ingesting diclofenac through feeding on the carcasses of treated animals. Even relatively small doses of the anti-inflammatory drug (LC50 = 0.1–0.2 mg/kg) were found to be toxic and caused kidney failure in the birds (Swan et al., 2006). Another commonly cited occurrence of effects on non-target organisms is the feminization of male fish and production of viable eggs following estradiol exposure, which has been linked to residue from oral contraceptives in freshwater (Kidd et al., 2007). At the invertebrate level, there is a wide variety of effects, physiological and behavioral, that can result from exposure to selective serotonin reuptake inhibitors (SSRIs), the most commonly prescribed class of antidepressant medications (Fong & Ford 2014). Both freshwater and marine species of gastropods exhibited foot detachment from substrates (Fong & Hoy 2012; Fong & Molnar 2013) whereas amphipod crustaceans altered their light preferences and activity levels (Guler & Ford 2010). In the present study, we found little effect at the behavioral level, but varied changes at the physiological level that targeted oxygen consumption and use.
Due to the variety of effects that can occur as a result of exposure to pharmaceuticals, risk assessment for this class of contaminants should include more than a single assay to assess impairment and to determine acceptable levels for discharge into the environment. Previous work has shown that environmentally relevant concentrations of naproxen dosed in flowing water are sufficient to induce changes in crayfish behavior (Neal & Moore 2017), however, similar concentrations used in this study produced no significant changes in behavior. This conflicting evidence suggests that one assay may not be sensitive enough to encapsulate the full scope of deleterious effects that this pharmaceutical can have on a non-target organism. Additionally, this evidence further supports that the mode of toxicant delivery plays a crucial role in shaping chemical exposure. As mentioned above, changes to behavior can arise from seemingly subtle changes to physiology (Kravitz 1988). For instance, modulating serotonin levels in decapod crustaceans is known to affect aggressive behavior (Kravitz 2000). One could hypothesize that exposure to pharmaceuticals that regulate serotonin release (SSRIs) could consequently impact aggressive behavior. A study that utilizes both a behavioral component that directly measures aggression, as well as a physiological measurement, such as tissue sampling for serotonin, would provide more information into this phenomenon than a behavioral assay by itself. By adding on the tissue sampling, one may be able to determine more exact concentrations at which aggressive behavior is significantly altered instead of approximations used for exposure treatments.
Moving forward, toxicity testing and environmental risk assessment of pharmaceuticals should seek to incorporate several levels of biological organization in their analysis as well as more realistic pulsed exposure patterns. Our results indicate that while an organism’s behavior may not significantly be altered by low levels of pharmaceuticals, acute exposure can still produce unexpected changes to physiology. By including more sensitive assays, we can more thoroughly elucidate the effects of more complex toxicants, such as pharmaceuticals and their mixtures, on non-target organisms. The standards used in toxicology today (LC50, EC50, NOEC, LOEC, etc.) are useful but limited in application, as the determining criteria for harm is mortality among test organisms. Additional standards that describe effects of toxicants in sublethal concentrations are one of several missing links in a comprehensive picture of chemical exposure.