We assessed the environmental risk of diclofenac sodium in European groundwaters using different scenarios. Overall, we found that the MECs of diclofenac sodium in European groundwaters in all the investigated scenarios of risk were consistently much lower than the NOEC of the groundwater-adapted asellid crustacean Proasellus lusitanicus. Indeed, P. lusitanicus exhibited significantly lower sensitivity to diclofenac compared to both surface and other groundwater species. This asellid species (adult mean body length: 5.2 mm) [4] was one order of magnitude more resistant to diclofenac sodium than the groundwater copepod Nitocrella achaiae Pesce, 1981 (adult mean body length: 0.5 mm) [22] and twice as resistant as the stygophile copepod Diacyclops crassicaudis crassicaudis (Sars G.O., 1863) (adult mean body length: 0.8 mm) [26]. Body size and metabolic rates likely play a role in determining such a sensitivity difference. The process of uptake involves the movement of diclofenac molecules from the surrounding water across the invertebrates’ body surfaces, such as their gills, exoskeleton, or integument [27]. The drug can passively diffuse through cell membranes due to its small size and lipophilic (fat-soluble) nature and be transported to tissues and organs [28]. Smaller-bodied organisms, such as copepods, have a greater surface-to-volume ratio, which causes a higher passive diffusion of diclofenac and other substances in comparison to larger species [29]. Once inside the invertebrate’s body, diclofenac reaches and affects tissues and organs. This process depends on the species’ metabolic rates, which serve as a proxy for their physiological rates. Notably, P. lusitanicus exhibits metabolic rates (86 ng O2/mg x h) [4] approximately one order of magnitude lower than those of groundwater copepod species (for instance, Moraria sp.: 913 ng O2/mg x h) [5]. This may result in a reduced uptake rate of diclofenac and subsequent internal transport in P. lusitanicus compared to smaller invertebrate species, particularly under sub-chronic exposure conditions. Similar delay in the uptake of organic compounds have been reported in other groundwater invertebrate species [30].
The environmental risk assessment conducted using the sensitivity of P. lusitanicus to diclofenac has yielded scenarios indicating no significant risk. These findings suggest that the current measured environmental concentrations of diclofenac in European groundwaters do not pose a substantial threat to the survival of this groundwater species. However, our study shows that the sensitivity of other groundwater species to diclofenac varies. The differing sensitivities of species within a given ecosystem can greatly influence the assessment of risks associated with specific contaminants. In the case of diclofenac, if the focus had been on the groundwater copepod species N. achaiae as the indicator species [22], the risk scenarios would have likely portrayed a higher level of concern and highlighted a greater potential risk to groundwater ecosystems. These observations emphasize the need for comprehensive assessments that take into account the sensitivities of multiple species within an ecosystem. A critical aspect regarding the ERA procedures is the recommendation to use three model taxa representing three trophic levels when determining the PNEC of pharmaceutical compounds [19, 21]. While surface water ecosystems typically feature algae, crustaceans and fish (primary photosynthetic producers, primary consumers and predators), in groundwater environments, primary production is limited to chemolithoautotrophic processes, if present [1]. As a result, these ecosystems heavily rely on the transport of organic matter from the surface [31]. The role of microorganisms in groundwater ecosystems is not fully understood, but they are believed to play a significant role in transforming organic matter, which can support entire food webs [32].
However, conducting ecotoxicological studies with groundwater species presents numerous challenges, as discussed in detail in Di Lorenzo et al. [21]. These challenges arise from the unique physiological characteristics of groundwater species, such as their low reproduction rates, long life spans, and low metabolism [33, 34, 35], which make them ill-suited for tests designed for surface water invertebrates [21]. Accessing groundwater habitats and collecting groundwater species require expertise and specific equipment, making the process more complex and time-consuming compared to sampling surface water organisms [7]. Taking into account all of these factors, employing surface water species as surrogates to estimate the sensitivity of groundwater species to chemical contaminants (upon applying appropriate correction factors that consider the specific traits and sensitivities of groundwater fauna), appears to be a reasonable and practical approach [22, 36]. The findings of this study highlight that the European guidelines [19, 20] for the environmental risk assessment of pharmaceutical compounds in groundwater present the most concerning environmental risk scenario of diclofenac. Based on Scenarios 1 and 2, it becomes evident that a significant number of European groundwaters are at risk from diclofenac contamination. These scenarios indicate that the presence of diclofenac in these groundwater systems poses a high risk at concentrations exceeding 5 ng/L. The implication arising from this is that the EQS for diclofenac in groundwater should be < 5 ng/L. This value may initially appear overly restrictive, especially considering the higher resistance observed in groundwater species like P. lusitanicus and N. achaiae. However, we believe that an EQS of 5 ng/L takes into account the need for precautionary measures to safeguard groundwater ecosystems, which are delicate and vulnerable [3, 7], and we will provide an explanation for this reasoning. First, it is important to acknowledge that pharmaceuticals frequently co-occur in groundwater [37, 38]. However, the potential effects of pharmaceutical mixtures on P. lusitanicus or groundwater fauna as a whole remain poorly understood [e.g., 39, 40]. It is possible that synergistic or additive effects may occur, which would justify the establishment of an EQS for diclofenac that is significantly lower than the PNEC for these species. Second, the results of ecotoxicological trials may not fully represent the potential effects of diclofenac sodium on the real populations of groundwater species. Our toxicity test specifically focused on the adult stages of the groundwater crustacean P. lusitanicus, while it has been observed that the sensitivity of juveniles to diclofenac can differ from that of adults in other crustaceans [26]. Studies on the epigean cyclopoid species D. crassicaudis crassicaudis have shown that juvenile stages are approximately twice as sensitive to diclofenac compared to adults [26]. This difference can be attributed to various factors, including the role of calcium in crustaceans. Calcium is essential for the mineralization of the new cuticle, and the pathway of calcium accumulation may inadvertently lead to the uptake of contaminants. The higher rate of molting and growth during the earlier stages of life makes juvenile crustaceans more susceptible to the toxic effects of substances like diclofenac [29]. Finally, the effects of diclofenac in more natural or semi-natural conditions (such as those reproduced in a mesocosm) may be more severe compared to what is observed in ecotoxicological trials where the diclofenac ingestion is not considered because the animals are not fed during the experiments.
Following this reasoning, the EQS for diclofenac in groundwater (5 ng/L) would be 10 times more restrictive than the current EQS in surface water (50 ng/L). This 10x difference appears to be a rule of thumb that will be incorporated into the new law revising the Water Framework Directive, the Groundwater Directive, and the Environmental Quality Standards Directive (Surface Water Directive) in Europe [41]. Notably, to enhance the protection of the EU's groundwater resources, members of the European Parliament have called for threshold values applicable to groundwater to be set at levels ten times lower than those established for surface water. This step means a significant commitment to safeguarding groundwater ecosystems and species.
One important aspect to consider in our study is the geographical bias of the depicted scenarios. The extent of monitoring for diclofenac sodium in European groundwaters varies significantly among different countries. The WATERBASE database, for instance, initially had limited information, with only one collection site in France in 2013. However, in 2019, more countries contributed to the database, with Italy, Slovakia, France, and the Czech Republic providing a substantial number of sample sites. It is also important to acknowledge that regulatory monitoring programs often focus on potentially problematic sites, which may not fully depict a realistic scenario. This selective monitoring approach might result in missing higher concentrations that could occur in specific locations, such as those near emission sources like hospitals.