The effects of naproxen sodium on the growth, reproduction, survival, and feeding of a freshwater pond snail

Animal housing and care

We purchased pond snails (Physa spp.) from Carolina Biological Supply Co. and held them in a temperature-controlled room at 23 °C with LED lights on a 12-hour cycle. Snails were fed Aquatic Arts Algae Wafers every 3 days (per food manufacturer directions) and their water was changed biweekly from premade solutions. Initially, snails were held in a tank of dechlorinated tap water, and then transitioned to Holtfreter’s solution over the first week. This solution was made of distilled water, sodium chloride (3.50 g/L), calcium chloride dihydrate (0.133 g/L), magnesium sulfate heptahydrate (0.205 g/L), all procured from Fisher Scientific, and sodium bicarbonate (Arm and Hammer, 0.200 g/L). We also supplemented with calcium carbonate (98%, Fisher Scientific, Hampton, NH, USA). We then allowed the snails to further acclimate in the tank for another week prior to the start of the experiment, where they were placed in individual glass mason jars filled to 0.3L with their experimental treatment solution, and the experiment began upon exposure.

Experimental methods

To test the effect of naproxen on the snail demographic rates (reproduction, growth, and survival), we conducted a month-long lab experiment where snails were housed in individual containers (one snail per jar) with four nominal levels of naproxen sodium (Fisher Scientific, Hampton, NH, USA) exposure in Holtfreter’s solution: 0 µg/L, 100 µg/L, 1,000 µg/L, and 10,000 µg/L, with 10 replicates for each treatment (n = 10). Naproxen is often sold in the form of naproxen sodium, as the salt version is easier to absorb (Brutzkus, Shahrokhi & Varacallo, 2025), and is often used to test the biological effects of naproxen in non-target organisms (e.g., Li et al., 2016; Kwak et al., 2018; Cory et al., 2019). Although higher than currently observed concentrations (107 ng/L, Ebele, Abou-Elwafa Abdallah & Harrad, 2017), our experimental levels (100–10,000 µg) reflect the range from prior studies of naproxen on fish (Ji et al., 2013) to levels in line with those found in human urine (Aresta, Palmisano & Zambonin, 2005). We were unable to verify the nominal concentrations, although previous studies note that naproxen is fairly stable in water over short time periods (Ji et al., 2013; Xu et al., 2019), with other studies finding a half-life of over one year (Toński et al., 2019).

Each level of naproxen had 10 replicates for each treatment (n = 10) for a total of 40 snails. Snails were haphazardly assigned to a jar, and each jar was randomly assigned to a treatment. The jars were randomly arranged in space in the temperature control room

We measured growth, reproduction, and survival weekly and feeding every 3 days. To measure growth, we measured the length of the snail (apex to base) with calipers and calculated a weekly growth rate (change in length). Physa spp. are hermaphrodites and will self-fertilize, so we measured reproduction by recording the number of egg sacs produced each week. Finally, we noted whether each snail was living or dead.

To measure the effect of naproxen on snail feeding, we placed 2 g of an algae aquarium food wafer (Aquatic Arts, Indianapolis, IN, USA) in each jar for four hours. At the end of the feeding period, we noted the presence or absence of a grazing scar.

Statistical analyses

We used a series of analyses to test the effects of naproxen concentration on snail demographics and feeding. We used generalized linear mixed effects models implemented in “glmmTMB” (Brooks et al., 2017) for snail growth, reproduction, and feeding. Only living snails were included in the analysis of growth, reproduction, and feeding. Because we were unable to verify the nominal concentration, we treated this value as a categorical variable, rather than a continuous concentration. For all models, week (continuous) and treatment (factor) were fixed effects along with their interaction and the individual snail was a random effect. We evaluated all models using the “DHARMa” package (Hartig, 2022), which included evaluating overdispersion. The model for snail growth assumed a Gaussian distribution and the validation process indicated a quadratic term on time was necessary for an adequate fit. Snail reproduction (number of egg sacs) assumed a Poisson distribution due to the count data, and feeding a binomial distribution (food consumed or not).

To analyze the survival data, we used a Cox proportional hazards model implemented in the “survival” package (Therneau, 2024), with post hoc analysis using the “survminer” package for pairwise comparisons (Kassambara, Kosinski & Biecek, 2016). All analyses were performed in R version 4.4.1 (R Core Team, 2024).

Discussion

Naproxen, an over-the-counter NSAID commonly used as a painkiller, is a common aquatic pollutant (Ebele, Abou-Elwafa Abdallah & Harrad, 2017). However, it might be preferable to other, more harmful NSAIDs, such as diclofenac, which has wide-reaching ecological and physiological effects (Näslund et al., 2020). In our experiment, we did not detect an effect of naproxen on growth (Fig. 1A) or reproduction (Fig. 1B), but naproxen did reduce survival (Fig. 1C). Additionally, snails exposed to our highest concentration (10,000 µg/L) of naproxen were less likely to feed than snails with lower levels of exposure (Fig. 2).

We found that at all tested levels of naproxen, the survival of Physa was reduced. By week 3, all exposure treatments were below a 50% survival rate, indicating that naproxen pollution could have large consequences for snail population sizes. Physid snails affect other members of the freshwater community through trophic interactions as prey for flatworms such as Dugesia lugubris (Tripet & Perrin, 1994), crayfish such as Procambarus simulans (Alexander & Covich, 1991), and fish such as Lepomis macrochirus. While much of this research has focused on the effects predators have on the snails (e.g., Goeppner et al., 2020), removing a key prey species has the potential to shift dynamics within the community.

Reductions in grazing due to naproxen pollution, either because of lower feeding or survival rates, could have cascading effects on the floral community and nutrient cycling (Bernot, 2013). Freshwater snails have large effects on their community through their grazing. Snail grazing reduces the amount and diversity of periphyton communities (Lowe & Hunter, 1988; Rezanka & Hershey, 2003) and the growth of macrophytes (Yang et al., 2020a). Given that predator exclusions can alter the periphyton community due to intense snail grazing (Martin et al., 1992), it is likely that reducing snail grazing due to pollutant presence would also affect community composition.

Many additional factors can affect the grazing behaviors of freshwater snails, such as parasite presence (Bernot & Lamberti, 2008), sediment loading (Kent & Stelzer, 2008), predator identity (Bernot & Turner, 2001), and water flow (Schössow, Arndt & Becker, 2016). In natural systems, snails will be subject to these factors in combination with pharmaceutical pollution, which could yield unpredictable results when pharmaceutical pollution can alter species interactions such as predation (Bose et al., 2022). Therefore, future work should include not only naproxen, but also naproxen in combinations with other abiotic and biotic stressors of Physa spp.

Additionally, while we were unable to detect an effect of naproxen on reproduction, we only counted egg masses. It is possible that instead of limiting the number of egg sacs produced, naproxen limits the hatching rate, the number of eggs in each sac, and/or affects the egg size. Reductions in hatching rates of Physa have been observed with other pollutants such as microplastics (Michler-Kozma, Neu & Gabel, 2022; Merbt et al., 2024). If this is also the case for naproxen and Physa, there could be additional negative ramifications on snail population dynamics, especially given the harmful effects of naproxen on survival.

One effect that was consistent throughout multiple response variables was the deterioration in snail health as the experiment progressed. Even with consistent feeding rates (Fig. 2), water changes, and calcium carbonate supplementation, snail demographic rates decreased throughout the experiment (Fig. 1), suggesting less than favorable conditions in their artificial habitats. Our experiment lasted longer than many studies on contaminants and Physa survival (e.g., 96 h (Gao et al., 2017)), and it is possible these small containers are not ideal for longer-term experiments. Given this result, it is possible the effects of naproxen were underestimated in this study due to this additional variability or overestimated if the effects of naproxen were exacerbated by laboratory conditions.

Pharmaceutical pollution can have large consequences on aquatic ecosystems (e.g., affect the recovery of a community after a disturbance, (Michelangeli et al., 2024), as well as neighboring terrestrial habitats (Previšić et al., 2021). The degree to which those effects propagate throughout the ecosystem can depend on the effects on freshwater molluscs. Pharmaceuticals can be passed up the food chain through freshwater snails (Yang et al., 2020b), similarly to how they transfer other pollutants, such as microplastics (Holzer et al., 2022), although the degree of transfer depends on the pharmaceutical. Previous studies have proposed naproxen as a preferred NSAID because it bioaccumulates less than alternatives such as diclofenac (Näslund et al., 2020)

Previous studies found harmful effects of NSAIDs such as ibuprofen (Pounds et al., 2008; Elías et al., 2025b), diclofenac (Bouly et al., 2022a) on mollusc growth. While we failed to detect a significant effect, our results (Fig. 1A) also trended in this direction. Some studies have noted complexities in the response of growth to NSAID exposure (Bouly et al., 2022b; Elías et al., 2025b), indicating the need to explore exposure over the entire development period, multiple generations, and in the presence of other contaminants. Variation also exists in the literature regarding the response of mollusc reproduction to NSAID exposure. While ibuprofen actually increased snail reproduction (Elías et al., 2025b), diclofenac, like our findings with naproxen (Fig. 1B), had no effect (Bouly et al., 2022a).

We found a harmful effect of naproxen on survival (Fig. 1C), which aligns with the effects of ibuprofen (Pounds et al., 2008). However, not all studies found a harmful effect (e.g., Bouly et al., 2022a). Given these disparate findings, it is likely that not only context and concentration, but the specific species play an important role in the response of freshwater molluscs to NSAID pollution. Future work is needed to compare the response of snails and other freshwater organisms to other NSAIDs to better understand the effects of NSAID pollution on freshwater communities.