Multigenerational exposure of Daphnia carinata to NSAIDs in individual concentrations and binary mixtures

doi:10.21203/rs.3.rs-4724417/v1

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Multigenerational exposure of Daphnia carinata to NSAIDs in individual concentrations and binary mixtures

https://doi.org/10.21203/rs.3.rs-4724417/v1

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Pharmaceuticals, including non-steroidal anti-inflammatory drugs (NSAIDs) like ibuprofen and naproxen, are widely used for medical purposes but have also become prevalent environmental contaminants. However, there is limited understanding of their effects on aquatic organisms, especially regarding multigenerational and mixture exposures. This study aimed to evaluate the toxicological impacts of ibuprofen and naproxen, individually and in combination, on three generations of Daphnia carinata, a freshwater organism. Daphnids were exposed to environmentally relevant concentrations of ibuprofen and naproxen (0.1, 0.5, 2.5 µg/L and 0.1 + 0.1, 0.1 + 0.5, 2.5 + 2.5 µg/L) throughout multiple generations. Various endpoints were assessed, including reproduction, body size, reproduction recovery, and behaviour. The results revealed that both ibuprofen and naproxen had negative impacts on reproduction, leading to reduced reproductive output across generations.Additionally, daphnids exhibited changes in body size, with significant alterations observed in the F2 and F3 generations. Male individuals and ephippium were also present at all concentrations throughout all generations. Although reproduction recovery could not be observed in daphnids after one generation in clean water, the average number of neonates was higher in a few treatments in generation F4 compared to generation F3. In addition, binary mixtures of the drugs showed synergistic effects on daphnids' reproduction. The multigenerational approach provided valuable insights into the long-term effects of these NSAIDs on reproductive success and population dynamics. This study contributes to understanding the ecotoxicity of ibuprofen and naproxen in aquatic organisms, particularly in a multigenerational context and in the presence of mixture exposures.

Non-steroidal anti-inflammatory drugs

reproduction

daphnia

environmentally relevant concentrations

ephippia

heart rate

feeding rate

Pharmaceuticals play a crucial role in treating diseases and have significantly improved the quality of life for many individuals. The pharmaceutical industry represents a rapidly growing market worldwide, with soaring sales and consumption rates (Peña et al., 2021). Consequently, the presence of pharmaceuticals in the environment has become increasingly widespread. These chemicals enter different ecosystems through various sources, including aquaculture, human activities, livestock, and the excretion of metabolites and residues (Alkimin et al., 2020; Zhou et al., 2019). Furthermore, due to the diverse mechanisms of action exhibited by different drugs, their presence in the environment poses challenges in detection and assessment and interferes with their removal in wastewater treatment plants and drinking water (Quesada et al., 2019). Despite being detected in the environment at very low concentrations, these drugs have been shown to induce changes in the life cycle (Grzesiuk et al., 2020; Jan-Roblero & Cruz-Maya, 2023; Zhao et al., 2024), metabolism (Fekadu et al., 2019; Zhang et al., 2020; Xu et al., 2019), and genetic pathways (Milan, et al., 2013; Mezzelani et al., 2018; Stancová et al., 2015) of aquatic animals.

Despite increased knowledge about the toxic effects of pharmaceuticals on a diverse range of animals and ecosystems, there are still a few gaps in our understanding of the topic. While it is expected to evaluate the effects of chemical compounds individually, organisms, especially aquatic species, are typically exposed to contaminants that vary in their components and concentrations (Faust et al., 2003). Therefore, assessing the effects of chemicals in individual concentrations and not mixtures may not reveal the real impacts they cause on the environment. Additionally, pharmaceutical mixtures often exhibit higher ecotoxicity than single chemicals alone (Gody & Kummrow, 2017), which can have compounded adverse effects on non-target organisms.

Non-steroidal anti-inflammatory drugs (NSAIDs) are currently the most prevalent contaminants of emerging concern found in the environment (Świacka et al., 2021). These drugs have analgesic and antipyretic properties and are widely used worldwide for human and veterinary purposes (Rocca et al., 2015; Parolini, 2020). Their mode of action involves inhibiting the function of cyclooxygenases (COX), enzymes involved in inflammatory processes, thereby affecting the biosynthesis of prostaglandins (citation missing). Mammals, including higher animals, have both forms of COX enzymes (COX-1 and COX-2), and as a result, NSAIDs affect them (citation missing). However, since invertebrates and other lower vertebrates also possess at least one isoform of COX, it is known that these pharmaceuticals can also impact these animals (Alkimin et al., 2020; Muñiz-González, 2021; Hayashi et al., 2008; Heckmann et al., 2007; Han et al., 2010; Milan et al., 2013; Li et al., 2016).

Ibuprofen and naproxen were chosen as the pharmaceuticals for our study because they are among the most commonly used NSAIDs worldwide. Additionally, their consistent detection in aquatic environments over the years is attributed to their widespread usage (Feng et al., 2021; Paíga et al., 2012; Suzuki et al., 2014; Ferrando-Climent et al., 2012; Jia et al., 2021). Therefore, considering the continuous release of these drugs into the aquatic environment, it is necessary to conduct prolonged exposure studies to assess better and comprehend the long-term effects of these chemicals on aquatic organisms (Dietrich et al., 2010). Multigenerational studies can provide more accurate data regarding an organism's sensitivity than single-generation studies, as they exhibit lower variance fluctuations. Furthermore, multigenerational studies evaluate an organism's life cycle under conditions similar to those in its natural habitat, which aids in understanding the toxic effects within a natural ecosystem (Araujo et al., 2019). While a few studies have confirmed the ecotoxicity of ibuprofen and naproxen to certain aquatic species, only a limited number have assessed the toxic effects of these NSAIDs using a multigenerational approach and considering pharmaceutical mixtures (Ogunbanwo, 2021; Melvin et al., 2014; Borgmann et al., 2007; Molina-Fernandez et al., 2016).

In light of this, there is a significant requirement for further understanding regarding the ecotoxicity of NSAIDs, both as individual substances and in combination, through multigenerational studies involving aquatic species. Therefore, this study aimed to assess the toxicological effects of ibuprofen and naproxen, individually and in combination, using a multigenerational approach with Daphnia carinata, a freshwater organism native to Australia. Furthermore, to our knowledge, no multigenerational studies have investigated the ecotoxicity of ibuprofen and naproxen in combination with Daphnia carinata.

2.1. Pharmaceuticals

Ibuprofen (Sigma-Aldrich, CAS Number: 31121-93-4) and naproxen (Sigma-Aldrich, CAS Number: 26159-34-2) were purchased from Sigma-Aldrich Australia. Both chemicals are water-soluble and have a purity of ≥ 98%. The stock solution was prepared separately in Milli-Q water, with 1000 mg of the NSAIDs each. Ibuprofen and naproxen chemical information and structure can be found in the supplementary information (Table S1).

2.2. Cladocerans

Daphnia carinata were maintained under controlled temperature (21 ± 1ºC), luminosity (150 µmol m-2s-1), and a photoperiod of 16:08 hours light to dark cycle. Stock cultures were kept in 400 ml beakers containing 300 ml of soft reconstituted water with a pH range of 6.0 to 9.0. The medium was renewed every two days (three times a week), with constant monitoring of the parameters. The animals were fed thrice a week with Raphidocelis subcapitata microalgae obtained from a suspension with a 5 x 10⁵ cells/mL concentration.

2.3. Experimental design- multigenerational exposure

The exposure concentrations were determined based on previous chronic studies with D. carinata and IBU and NPX, as well as the concentrations of both NSAIDs found in the aquatic environment (Ebele et al., 2017; Wojcieszyńska & Guzik, 2020). Consequently, no acute toxicity test was conducted before initiating this multigenerational study. The multigenerational test followed OECD test guidelines 211 (OECD, 2012).

Daphnia carinata neonates were exposed to three single concentrations of IBU and NPX individually (0.1, 0.5, 2.5 µg/L) and to four binary mixtures (0.1 IBU + 0.1 NPX, 0.1 IBU + 0.5 NPX, 0.5 IBU + 0.1 NPX, 2.5 IBU + 2.5 NPX µg/L). The culture medium was renewed every 48 hours, and water quality parameters were recorded before and after each change until the conclusion of the reproductive test. Daphnids of generation F0 were exposed to the NSAIDs for 21 days. Neonates collected from each concentration starting the 15th day were used to initiate the next generation. The test continued until the third generation (F3), after which neonates from F3 were reintroduced to ASTM water to assess crustacean recovery rates. The experiment spanned three months in total.

Endpoints assessed included survival rate, time to first brood, total number of offspring in the first brood, total number of neonates per female, adult daphnid size at the end of each generation, and immobile or dead neonates. Dead parent daphnids were excluded from statistical analysis.

2.4. Reproduction recovery

The reproduction recovery analysis was conducted according to OECD Test Guideline 211 (2002). Offspring from the F3 generation was exposed to a clean medium without pharmaceuticals to assess the reproduction of the daphnids (refer to Fig. 1).

On the 15th day of generation F3, neonates were randomly selected and transferred to glass vessels containing a clean medium for 21 days. The clean medium and algae were renewed every two days, following the protocol of the multigenerational exposure. These test vessels were maintained in a temperature-controlled room (21 ± 1°C) with a photoperiod of 16:8 hours (light: dark).

2.5. Binary mixture toxicity assessment

The Toxic Unit (TU) model is a robust and valuable tool for evaluating mixture toxicity, as it aggregates the toxic contributions of each component in the mixture (Woods et al., 2002). However, due to the investigation of only three combinations of mixtures within a multigenerational scenario, limitations arose in calculating dose-response endpoints such as EC20 and EC50. Therefore, a modified approach to the Toxic Unit (TU) was adopted in this study to assess the binary mixture toxicity of the following treatments: 0.1 + 0.1 (IBU + NPX), 0.1 + 0.5 (IBU + NPX), 0.1 + 0.5 (NPX + IBU), and 2.5 + 2.5 (IBU + NPX) µg/L. The total number of neonates produced over 21 days was converted into percentages (Supplementary Table 2). Subsequently, the percentage of the total number of neonates for each binary mixture in each generation (F0 to F3) was divided by the percentage of the total number of neonates for the individual concentrations of IBU and NPX (0.1, 0.5, and 2.5 µg/L) for each respective generation, as given in Eq. 1:

Saying this, when TU equals 1, the effect of the mixture is considered additive. If TU exceeds 1.2, it indicates an antagonistic effect (less than additive), while if TU is less than 0.8, it suggests a synergistic effect (more than additive) (Markovic, et al., 2021).

3.0. Statistical analysis

The reproduction data obtained from the experiment results were evaluated for normality using the Shapiro-Wilk test and for homogeneity of variances using Levene's test. When the results met the criteria for normality and homogeneity, they were subjected to One-Way ANOVA followed by Tukey's post hoc test at a 95% confidence level to identify significant differences among the NSAID treatments and the control group. However, if the normality assumption of the data was violated, the Kruskal-Wallis test with Bonferroni correction for post hoc analysis was performed instead. To analyse reproduction recovery, a normality test followed by the non-parametric Kruskal-Wallis test was conducted to assess differences between the F3 and F4 generations. All statistical analyses were performed using GraphPad Prism 10.2.0.

4.2. Multigenerational exposure of Daphnia carinata

4.2.1. Reproduction traits

No significant differences in survival percentage and the rate of reproduced daphnids were observed in all generations (F0-F3) exposed to IBU, NPX, and the binary mixtures compared to the control group (refer to Tables 1, 2, and 3). In generation F0 daphnids, there were no significant differences in the time of first reproduction, time of first egg production, total number of broods per individual, and total number of offspring per individual across all concentrations of IBU, NPX, and binary mixtures (assessed using Kruskal-Wallis Independent Samples with Bonferroni correction, P > 0.05).

Throughout generations F0 to F3, the control group released an average of 75.67 neonates per female. A concentration-dependent pattern for the mean number of neonates was observed with IBU and NPX across generations F1 to F3, showing significantly lower numbers of offspring at concentrations of 0.1, 0.5, and 2.5 µg/L compared to the control. Notably, concentrations of 0.1 µg/L IBU in generation F2 and 0.1 µg/L NPX in generation F2 did not exhibit a significant decrease in the cumulative mean number of neonates compared to the control (Kruskal-Wallis Independent Samples with Bonferroni correction, P > 0.05). Concerning the treatments involving binary mixtures, all except the 2.5 + 2.5 µg/L concentration showed a lower mean number of offspring from generation F1 to F3 compared to both the control group and the cumulative number observed in daphnids exposed to single concentrations of the pharmaceuticals (Kruskal-Wallis Independent Samples with Bonferroni correction, P < 0.05). Daphnids exposed to single concentrations of IBU and NPX exhibited a delay in the time of first egg production compared to the control (refer to Tables 2 and 3). In generations F2 and F3, a significant increase in the time of first egg production was observed in the IBU treatments, while the same effect was noted for NPX treatments in generations F1, F2, and F3. Regarding the drug mixture, all four treatments showed a delay in the time of first egg production compared to the control group in nearly all generations.

Table 2 Reproduction traits (mean + SE) of D. carinata exposed to ibuprofen for three generations.

We observed a distinct pattern in the cumulative mean number of neonates produced per female with single concentrations of IBU and NPX (0.1, 0.5, and 2.5 µg/L), showing a fluctuating trend across generations. IBU concentrations of 0.1 and 0.5 µg/L exhibited a decrease in the number of offspring from generation F0 to F1, followed by an increase at generation F2 and then a reduction in generation F3. Exposure to the single concentration of IBU at 2.5 µg/L showed a decrease in the number of offspring from generations F0 to F2, followed by an increase in generation F3. Similarly, NPX single concentrations showed a decline in the mean number of offspring from generations F0 to F2, followed by an increase in generation F3 (refer to Table 3). However, the binary mixtures exhibited a different effect than single concentrations. Binary concentrations of 0.1 + 0.1, 0.1 + 0.5 (IBU-NPX), and 0.1 + 0.5 µg/L (NPX-IBU) showed consistently fewer offspring across all generations (F0 to F3). Conversely, at the binary concentration of 2.5 + 2.5 µg/L, the mean number of neonates decreased from F0 to F2 and increased in generation F3.

In contrast, the total number of broods per individual showed little effect from single and binary treatments of IBU and NPX. No significant statistical differences were observed for the single concentrations of IBU compared to the control in the total number of broods per female at any generation (Kruskal-Wallis Independent Samples with Bonferroni correction, P > 0.05). For NPX single concentrations, significant differences were observed at generation F2 for concentrations of 0.5 µg/L (1.2 ± 0.54) and 2.5 µg/L (1.37 ± 0.32) compared to the control group (5.2 ± 0.27), as well as for three binary treatments (0.1 + 0.1; 0.1 + 0.5- IBU + NPX; 0.1 + 0.5- NPX + IBU) at the same generation. Significant differences were observed at generation F3 in daphnids exposed to the two binary treatments of 0.1 + 0.5 (IBU + NPX) and 0.1 + 0.5 (NPX + IBU) µg/L compared to the control group.

Age at first reproduction was significantly affected by both drugs in single and binary treatments. Daphnids exposed to single concentrations of IBU exhibited significant responses in generations F2 and F3, while those exposed to NPX single concentrations showed significant responses in generations F1, F2, and F3. Significant responses were also observed in generations F1, F2, and F3 for daphnids exposed to binary mix treatments.

Table 3 Reproduction traits (mean + SE) of D. carinata exposed to naproxen for three generations.

The symbol * represents statistically significant differences between the NSAID-exposed daphnids and the control (p < 0.05).

All the daphnid reproductive traits analysed concerning the individual concentrations of IBU and NPX were significantly different from those in the binary treatments in at least one generation (refer to Table 4).

Table 4. Reproduction traits (mean + SE) of D. carinata exposed to ibuprofen for three generations

Asterisks indicate statistically significant differences in exposed daphnids compared with the control group of each generation (p < 0.05).

4.2.2. Daphnia carinata body length

The body length of female adults at the end of the 21 days was significantly different from the control group in the F0 generation at 0.1 + 0.1 µg/L and the F1 generation in 0.5 NPX and 2.5 + 2.5 µg/L treatments (refer to Fig. 2A and Fig. 2B). In contrast, in generations F2 and F3 a significant reduction in body length was observed in all individual and mixture treatments when compared to the control group (refer to Fig. 2C and 2D).

Figure 2A. Body length of female (mean ± SE). Daphnia carinata exposed to single and binary mixtures of IBU and NPX in generation F0 after 21 days. A: single IBU, B: single NPX and C: binary mixtures. Asterisks indicate statistically significant differences from the control group (p < 0.05).

Fig. 2C. Body length of female (mean ± SE) Daphnia carinata exposed to single and binary mixtures of IBU and NPX in generation F2 after 21 days. A: single IBU, B: single NPX and C: binary mixtures. Asterisks indicate statistically significant differences from the control group (p < 0.05).

Figure 2D. Body length of female (mean ± SE) Daphnia carinata exposed to single and binary mixtures of IBU and NPX in generation F3 after 21 days. A: single IBU, B: single NPX and C: binary mixtures. Asterisks indicate statistically significant differences from the control group (p < 0.05).

4.3. Behavioural effects

Ephippia were absent in the control group. However, all treatments, with the exceptions of 0.1 µg/L IBU in generation F2 and 0.1 + 0.5 µg/L NPX + IBU in generation F3, exhibited the formation of ephippia across all generations (see Fig. 2C). Additionally, all treatments, except for the control group, produced male offspring from generation F0 to generation F3 (see Fig. 2A). Males and females displayed distinct morphological characteristics. Males are characterised by a smaller body size and a different ventral opening of the carapace (Martin-Creuzburg et al., 2018; Bethesda, 2005). They also had larger antennules (indicated by red arrows in Fig. 2A and B) and a modified post abdomen armed with a hook used during mating (Bethesda, 2005).

4.4. Post-exposure assessment

After three generations of exposure to the NSAIDs, the offspring of generation F3 was exposed to clean water for 21 days for the assessment of reproduction. After comparing all treatments of F4 generation to the control, we found that all treatments, except 2.5 + 2.5 µg/L, exhibited significant differences (p < 0.05) in comparison to the control group when comparing the total number of neonates (Fig. 3A). In addition, time of first egg production, time of first reproduction and total number of broods per individual exhibited significant differences (p < 0.05) from the control group in generation F4 (Supplementary Table 3).

The total average number of neonates in generation F4 for daphnids in the following treatments exceeded those observed in generation F3 for the same treatments: 0.1 and 0.5 µg/L IBU, 0.1 µg/L NPX, 0.1 + 0.1, 0.1 + 0.5 (IBU + NPX), and 0.1 + 0.5 (NPX + IBU) µg/L (see Supplementary Table 4). However, no significant differences were found by a Kruskal-Walli's test (p > 0.999) when comparing the total number of neonates between generations F3 and F4 (refer to Fig. 3B). Additionally, the time of first egg production in daphnids exhibited significant differences between generations F3 and F4 at all single concentrations of IBU and at 0.1 µg/L of NPX. In contrast, the time of first reproduction and the total number of broods per female did not differ.

Figure 3A. Total number of offspring of F4 generation in daphnids exposed to mixtures of IBU and NPX in comparison to the control group after 21 days. A: single IBU, B: single NPX and C: binary mixtures. Asterisks indicate statistically significant differences from the control group (p < 0.05).

4.5. Binary mixture assessment on reproduction

Based on the daphnid's reproduction at the end of 21 days over four generations, the chemical compound interactions between two drugs, IBU and NPX, were assessed using the toxic unit approach (Table 5).

Table 5 Toxic Unit (TU) model for assessing the effects of IBU and NPX binary mixtures on Daphnia carinata reproduction.

A synergistic action on D. carinata reproduction of the binary mixtures of IBU and NPX was indicated when the result was analysed using the Toxic Unit (TU) approach across all generations in the following mixtures: 0.1 + 0.1 (IBU + NPX) with 0.1 NPX as an individual concentration, and 0.1 + 0.5 (NPX + IBU) with 0.5 IBU and 0.1 NPX as individual concentrations. A combination of synergistic and additive actions was observed in mixtures such as 0.1 + 0.1 (IBU + NPX) with 0.1 IBU as an individual concentration, 0.1 + 0.5 (IBU + NPX) with 0.1 IBU as an individual concentration, 0.1 + 0.5 (IBU + NPX) with 0.5 NPX as an individual concentration, and 2.5 + 2.5 (IBU + NPX) with 2.5 IBU and 2.5 NPX as individual concentrations. Two antagonistic actions were observed: at generation F0 for daphnids in the binary mixture of 0.1 + 0.5 (IBU + NPX) with 0.5 NPX as an individual concentration and at generation F2 in the treatment of 2.5 + 2.5 (IBU + NPX) with 2.5 NPX as an individual concentration.

Although the removal rate of IBU and NPX can be up to 99% in wastewater treatment plants (Elmouatezz Bellah Kermia et al., 2016; Wojcieszyńska & Guzik, 2020; Kodom et al., 2021), the constant use of pharmaceuticals causes them to enter and contaminate the aquatic environment persistently (Gworek et al., 2019). Consequently, concerns have been raised about the potential consequences of pharmaceuticals reaching the environment, as these compounds can bioaccumulate and cause harmful effects on organisms due to chronic exposure (Rede et al., 2019). Non-steroidal anti-inflammatory drugs (NSAIDs) are in high demand worldwide, accounting for more than 15% of all drug groups detected in the environment (Świacka et al., 2021). However, data regarding the toxic effects of NSAIDs on lower species, such as invertebrates, are not substantial (Świacka et al., 2021). Our study contributes to the analysis of the effects of NSAIDs on aquatic organisms, particularly daphnids. Daphnids are essential organisms in freshwater ecosystems, playing a pivotal role in the food web by filtering smaller organisms and microalgae, thus helping control their numbers and composition (Jurgens, 1994). Observing that reproductive parameters of the daphnids were affected after one generation at environmentally relevant concentrations and when exposed to binary mixtures of pharmaceuticals in our present study, we inferred that the aquatic trophic structure could be modified and may suffer significant hazardous effects.

It is, therefore, evident that these drugs have delayed toxicity, emphasising the importance of multigenerational studies and the examination of binary mixtures of pharmaceuticals to detect continuing environmental effects.

5.1. Survival and exposure conditions

In generations F0 to F3, survival rates ranged between 90–100% in the control groups and 70–100% in IBU and NPX single treatments and binary mixtures (Tables 1, 2 and 3). The mortality rate was considered acceptable since it was under the OECD Test Guidelines (2011) for daphnia chronic test.

5.2. Multigenerational assessment

Multigenerational tests are crucial assessments of the toxic effects of chemical compounds on environments due to higher sensitivity at a population level, in comparison to generation tests, and due to their capacity to represent more realistically natural scenarios of exposure (Nederstigt, et al., 2022). However, despite their importance, long-term tests of several generations are infrequently carried out (Nederstigt, et al., 2022).

A few multigenerational studies have demonstrated significant differences in reproductive and/or morphological traits of invertebrates exposed to NSAIDs. To our knowledge, few multigenerational studies, specifically with freshwater invertebrates, assess the effects of NSAIDs. Therefore, some studies that support ours involve invertebrate organisms in general. In a recent study by Prud'homme et al. (2017), Aedes aegypti mosquitoes were exposed to 1 µg/L of IBU over six generations, resulting in increased mortality during the development stage and an accelerated development rate in the offspring of generation six. A sex ratio imbalance was also observed, with female mosquitoes representing most of the population. Another study by Prud'homme et al. (2018) found that chronic exposure of Aedes Aegypti mosquitoes to environmentally relevant concentrations of IBU did not affect lifespan or development in the F0 generation, but survival and time to develop were impacted in the following generation (F1). These results are similar to our study's, in which the F0 generation was slightly affected. Still, effects on reproductive parameters were observed in subsequent generations following exposure to IBU at environmentally relevant concentrations.

In a study by Grzesiuk et al. (2020), the effects of IBU on D. magna were assessed, and results comparable to those of our study were obtained. The daphnids were exposed to an environmentally relevant concentration of 4 µg/L of IBU for five consecutive generations. Although no significant differences were observed for most life history traits, including age and size at first reproduction, number of first clutch eggs, and individual growth rate, the study revealed morphological changes in the offspring, providing unique information. The first generation showed up to 70% of deformed offspring, while the fifth generation showed up to 90%. Among the observed morphological deformities, undeveloped embryos, lack of eyes, and abnormal antennules were the most significant and discussed findings. While our studies did not analyse deformities in daphnia embryos, we observed a decrease in the numbers of neonates throughout the generations, indicating that IBU plays a toxic role in daphnia offspring. Additionally, it is possible that if we had continued the analysis over other generations, similar effects may have been observed in subsequent generations. It is difficult to determine at what generation the subsequent effects cease since maintaining cultures for multiple generations is costly in terms of time and effort; however, it should be considered in future studies with D. carinata. Other multigenerational studies have also documented reproductive impairments at environmentally relevant concentrations. In the Dietrich et al. (2010) study, daphnids were exposed to a concentration of 0.36 µg/L over five generations. The results showed a delay in age at first reproduction in the F0 and F2 generations, an increase in the body length of neonates in the F1 and F5 generations, and an increase in the number of offspring in the F1 generation.

Also, in a study conducted by Bouly et al. (2022), the effects of diclofenac (DCF), a pharmaceutical belonging to the NSAID family, were assessed on the offspring of the freshwater gastropod Lymnaea stagnalis across two generations. The snails were exposed to different concentrations of DCF (0.1, 2, and 10 µg/L). The results showcased an interesting influence of DCF on critical aspects of snail development. In the F1 generation, snail growth and shell sizes were affected by DCF. An increase in shell size was observed at 0.1 µg/L, while a decrease in shell size was observed at 2 µg/L and 10 µg/L. The adverse effects were also extended to food intake rates, exhibiting a reduction in egg masses. However, in the F2 generation, the snails displayed a shorter hatching time, increased time to growth, enhanced food intake, and greater production of egg masses. These findings correlate with the results in our study, where daphnids exposed to both IBU and NPX had a significant impact on the growth of daphnids and the number of neonates produced per female across multiple generations, especially in the cases of binary mixtures exposure, indicating that both IBU and NPX, whether in single or mixed concentrations, had a detrimental effect on the reproductive capacity of the daphnids throughout multiple generations.

Prostaglandins play a pivotal role in the inflammatory process, and we can link this role to the decrease in the reproduction of daphnids observed in our study. Prostaglandins (PGs) are a fundamental class of lipids with hormone-like effects, playing a crucial role in various bodily systems. They support essential homeostatic functions, regulating animals' nervous, respiratory, cardiovascular, reproductive, endocrine, and immunological functions (Gad, 2014; López-Doval et al., 2020). Moreover, PGs are key regulators of the inflammatory response. The precursor of the PGs is arachidonic acid (ARA), an essential fatty acid (Davies, 2008). While the pathway of the metabolic transformation of ARA to PGs is well known in mammals and it is mediated by the cyclooxygenase enzyme (COX), lipoxygenase (LOX) and cytochrome P450 (Fig. 6), it remains an area of limited understanding in invertebrates (Garavito & Mulichak, 2003; Ferreira Barletta et al., 2020).

In essence, non-steroidal anti-inflammatory drugs (NSAIDs) work by inhibiting the activity of COX-1 or COX-2, isoforms of the cyclooxygenase enzyme (Bindu et al., 2020). These enzymes produce various PGs essential in the inflammatory response (Bindu et al., 2020). By blocking the function of these enzymes, NSAIDs prevent the transformation of ARA into prostaglandins, reducing inflammation and alleviating pain (López-Doval, et al., 2020). However, as these pollutants are blocking the action of COX enzymes, they could also be interrupting reproduction mechanisms associated with the enzyme (Heckmann, et al., 2008). Our findings align with these insights, as while the pathway of PGs in invertebrates remains less understood compared to mammals, a few studies suggest that PG synthesis between both groups could relate (Stanley & Kim, 2019; Ahmed & Kim, 2020). In a study by Heckmann et al. (2008), genes coding for COX and LOX enzymes but with more straightforward structures have been identified in Daphnia magna, indicating that NSAIDs could also play the same role in a diverse range of parameters, as in the mammals. We observed decreased neonate numbers across successive generations, indicating potential interference with reproductive mechanisms associated with blocking COX enzyme activity. Understanding the crucial role of PGs in the reproductive and immune systems of both mammals and invertebrates allows us to establish a direct correlation between the impact of NSAIDs and the vital parameters of these organisms.

Although the studies conducted by Prud'homme (2018 and 2017) and Grzesiuk (2010) mentioned above did not report statistically significant differences in life-history parameters, their findings are not to be dismissed. These studies highlight a common theme of reproductive effects of exposure to environmentally relevant concentrations of NSAIDs at different levels, ultimately impacting life trait parameters. Although the exact mode of action of NSAIDs in aquatic organisms that leads to harmful effects in their reproduction is still not well understood, a few authors shared their results alongside their hypothesis. According to Choi et al. (2022) and Heckmann et al. (2008), the impact of NSAIDs on the synthesis of COX enzymes can disrupt oogenesis and impair reproduction. The interference with vitellogenesis and steroidogenesis, both closely linked to sex hormones and organism development (Kwak et al., 2018; Gonzalez-Rey & Bebianno, 2012), has been proposed as potential mechanisms for reproduction impairment. However, the effects of NSAIDs on the enzyme families (COX, LOX, and CYP450) involved in eicosanoid biosynthesis may vary among different animal groups, as observed by diverse responses in aquatic organisms (Han, et al., 2010). This highlights the need for further investigations to understand the specific effects of NSAIDs on eicosanoid biosynthesis.

Our research findings further corroborate these observations, as we observed toxic effects of both IBU and NPX, even at the lowest concentrations, on the reproductive traits of daphnids for three consecutive generations (F1 to F3). In addition, previous studies assessing the impact of NSAIDs on aquatic organisms across a single generation suggested that these pollutants could interfere with eicosanoid synthesis, causing further hazardous effects on the reproduction of aquatic organisms (Han et al., 2010; Du et al., 2016). Thus, even low levels of NSAIDs in the environment may harm the reproductive success of invertebrates, potentially affecting communities, food chains and overall ecosystem function.

A few other studies presented a different approach when explaining the effects observed from generation to generation. According to Tsui & Wang (2004), Lam & Wang (2009), LaMontagne & McCauley (2008), and Vandegehuchte et al. (2010), the primary effects of multigenerational exposure to chemicals can be attributed to two mechanisms: maternal transfer or transgenerational exposure. While the exact mode of action for maternal transfer is not fully understood, it is known to transmit information from parents to offspring in a non-genomic manner. On the other hand, transgenerational exposure involves the transfer of epigenetic marks through the germline without altering the primary structure of DNA (Hao, 2014; Costello & Schones, 2018). in a study conducted by Baker et al. (2013) using zebrafish, significant reproductive changes were observed in the offspring after one generation after exposing the eggs of the F0 generation to sublethal levels of pollutants, suggesting that multigenerational exposure to contaminants during the developmental stage can have harmful effects on subsequent generations and their offspring.

Another intriguing and comparable finding in our study, which aligns with the investigation by Bouly et al. (2022) and Dietrich et al. (2010), is the observed pattern of a decrease in offspring number in one generation followed by an increase in the subsequent generation. Furthermore, the study by Dietrich et al. (2010) reinforces an additional finding in our research. In both studies, after exposure to NSAIDs across multiple generations, a delay in the age of first reproduction was observed. In our study, all single concentrations of IBU, NPX and binary mixtures displayed a delay in the age of first reproduction in F1 and F2 generation compared to the control. At all NPX doses and 2.5 + 2.5 µg/L and 0.1 + 0.1 µg/L, the daphnids reached maturity sooner than the F2 generation. As Bouly et al. (2022) suggested, the increased offspring production and acceleration of reproductive maturity age could indicate a fecundity compensation mechanism. This phenomenon suggests that organisms may employ this strategy, allocating more resources to reproduction to offset the detrimental effects on their life cycle (Minguez, et al., 2015).

Furthermore, Lei et al. (2022) highlighted a rise in the offspring count, coupled with a reduction in the body growth of the daphnids following exposure to prevavastin, a cholesterol-lowering medication. We observed a similar trend in our study. In line with Lei and collaborators' findings, this event may be attributed to energy allocation towards detoxification processes and survival rather than maintenance and growth under stressful conditions.

5.2.1. Presence of males and ephippia

In our study, males and dormant eggs were consistently observed across various concentrations of IBU, NPX, and their binary mixtures throughout all generations. These findings align with previous research by LeBlanc et al. (2005), who highlighted the crucial role of juvenile hormones in regulating reproduction and development in daphnids, including the production of males. Moreover, Tatarazako et al. (2003) demonstrated that exposure to juvenile hormones and their analogues resulted in an increased production of males in daphnids, suggesting a potential link to the trade-off mechanism from asexual to sexual reproduction.

The presence of daphnids and their reproductive activities play a significant role in the freshwater ecosystem by serving as a crucial link in the energy transfer between primary and secondary producers, contributing to the regulation of algae and the microbial community, thereby exerting important ecological control (Dodson & Hanazato, 1995; Miner et al., 2012; Wojtal-Frankiewicz, 2012). Non-sexual reproduction is generally regarded as an efficient mechanism, as it avoids the costs associated with male production and allows for the conversion of more resources into offspring (Gerber, et al., 2017). However, in the case of daphnids, which exhibit a cyclic life cycle involving both sexual and asexual reproduction, there are costs involved, as females can perform only one reproductive mode at a time, leading to a trade-off between the benefits of each strategy (Gerber, et al., 2017). This interplay between sexual and asexual reproduction in daphnids reflects the inherent complexities of reproductive optimisation, where organisms must choose strategies to ensure the survival and success of the next generation.

Under optimal environmental conditions, daphnids typically exhibit an asexual life cycle known as parthenogenesis, where the offspring are predominantly female (Eads, et al., 2008). However, in response to specific environmental triggers such as limited food availability, overcrowding, turbulent waters, or droughts can induce the development of males from the eggs, thus enhancing the reproductive fitness of the population (Wuerz et al., 2019; Eads et al., 2008). Stressful conditions also contribute to forming ephippia, which are sexual eggs that remain dormant until the habitat conditions become favourable again for optimal reproduction (Abrusán et al., 2007). However, this physiologically demanding process of sexual reproduction incurs costs for daphnids (Lynch, 1983), and the production of dormant eggs disrupts rapid population growth (Zhang et al., 2016). The presence of ephippium and male offspring in the populations exposed to both NSAIDs, both individually and in binary mixtures, unequivocally demonstrated a notable shift from asexual to sexual reproduction in the tested groups of D. carinata. This shift signifies an adaptive response to the environmental stressors imposed by the NSAIDs, as the organisms employ sexual reproduction as a survival strategy to ensure the long-term viability of their population. Thus, a shift might have happened when D. carinata was exposed to the NSAIDs in our study.

While recent studies in the literature did not report the presence of males or ephippium in daphnid populations when exposed to NSAIDs, investigations with Daphnia magna and pesticides have revealed that these pollutants act as juvenile hormone analogues, leading to a decrease in offspring numbers and the induction of male production (Oda et al., 2005; Ginjupalli & Baldwin, 2013; Olmstead & LeBlanc, 2003). Interestingly, studies by Heckmann et al. (2008) demonstrated that ibuprofen induced juvenile hormones in D. magna without observing male production. These findings suggest that IBU may mimic the effects of juvenile hormones in daphnids, potentially leading to an increase in male production.

5.3. Toxic Units Mixture Assessment

Both NSAID drugs examined in this study are known to share the same mode of action in mammal models, as documented by Manrique Moreno et al. (2009). These pharmaceuticals inhibit two enzymes, cyclooxygenases 1 and 2 (COX-1 and COX-2), which are involved in prostaglandin synthesis. Prostaglandins, a group of lipids with hormone-like functions, contribute partially to the onset of inflammatory and pain processes (O'Day & Graham, 2013). When compounds share a common mechanism of action, pharmaceutical mixtures are generally expected not to interact, resulting in additive toxicity (Markovic et al., 2021). However, in our study, the binary mixture experiments revealed synergistic and additive effects, with more synergistic events observed in most treatments across all generations.

While no significant differences (p < 0.05) were observed when comparing the treatments of individual pharmaceuticals to the binary mixtures, bar graphs revealed that D. carinata produced lower neonate numbers when exposed to the binary mixtures. Similar outcomes were observed in several studies in the literature. In a study by Cleuvers (2004), Daphnia magna and D. subspicatus exposed to NSAID mixtures of diclofenac, ibuprofen, naproxen, and aspirin in concentrations ranging from 2.5 to 241.9 mg/L showed higher toxicity levels than those exhibited by the individual drugs. Another study by Cleuvers (2008) exposed Daphnia magna to an NSAID mixture of ibuprofen, diclofenac, and naproxen in concentrations ranging from 5 to 64 mg/L, revealing a higher rate of reproduction inhibition in the mixture treatment compared to the individual pharmaceuticals. Additionally, a study on NSAID mixtures on Daphnia magna evaluated the binary mixture of ibuprofen and indomethacin at 1 mg/L, showing a more significant decrease in feeding rate when exposed to the binary mixture compared to the concentration of pharmaceuticals individually (Michalaki & Grintzalis, 2023).

Synergistic effects between NSAIDs had also been observed in mammals when these drugs were administered together (Miranda et al., 2006; Miranda et al., 2009), which can also relate to the findings in our study.

5.4. Recovery assessment

When comparing all treatments of the F4 generation to the control group, we observed significant differences (p < 0.05) for all parameters assessed, inferring no recovery occurred after exposure to NSAID concentrations. However, following the exposure period to clean water, we observed an increase in the number of neonates in the single concentrations of IBU and binary mixtures when comparing the F3 and F4 generations, leading us to suppose that recovery is occurring slowly.

Nevertheless, there was no increase in the number of offspring in organisms from exposure to single concentrations of NPX (generation F4), except for concentration 0.1 µg/L. It is important to note that NSAIDs interfere with COX synthesis, and these enzymes play several roles in animals, including fertility functions. These roles may be linked to the control of reproduction not only in vertebrates but also in invertebrates, involving processes such as egg-laying, oogenesis, ovulation, vitellogenesis, and gonad maturation (Murdoch et al., 1993; Spaziani & Hinsch, 1997; Zahradnik et al., 1999; Stanley, 2006; Schlotz et al., 2012; Ahmed et al., 2018). Thus, this could assist in explaining why the recovery in reproduction observed in the tested daphnids occurs when they are no longer exposed to this pollutant, even at a slow pace.

Several studies have also reported a recovery mechanism following exposure to IBU, similar to our research findings. These studies observed a subsequent depuration phase after IBU exposure, leading to the restoration of normal physiological functions. For instance, in a study by Trombini et al. (2019), after exposing the marine clam Ruditapes philippinarum to diclofenac, ibuprofen, and carbamazepine for 14 days, biochemical changes were induced in the organisms. However, after 7 days of exposure to a clean medium, the clams could cope with the stress, showing recovery of their metabolic responses. Similarly, in another study by Trombini et al. (2021), crayfish (Procambarus clarkii) could recover their biochemical responses and gene expression functions after discontinuing exposure to IBU concentrations.

Furthermore, in a study by Heckmann et al. (2008), D. magna was exposed to IBU concentrations for 10 days, followed by 10 days of exposure to clean water. The results showed that during the exposure period, there was a decrease in the number of offspring produced. However, during the recovery period, the organisms exhibited accelerated reproduction and reached a population size equivalent to that of the control group. Heckmann et al. (2008) also discussed the results of a newly proposed recovery mechanism that could also apply to the recovery results observed in our study, shedding light on this mechanism and providing further insights into the potential factors influencing the recovery of reproductive functions after exposure to IBU. According to the investigators, daphnids could increase fecundity as a compensatory response to reproduction reduction during stressful periods. Drawing a parallel with the phenomenon of compensatory fecundity and increased body size, where after a period of stress, animals would allocate their energy budget to body growth instead of reproduction, Heckmann and colleagues propose a similar concept known as "catch-up reproduction". This concept suggests that after a period of reproduction inhibition, offspring production would increase when animals are more susceptible and the opportunity for reproduction is available within a specific timeframe.

No similar findings regarding the non-recovery effect of daphnids from the F4 generation resulting from single concentrations of NPX were found in the existing literature. Despite NPX being a widely used non-steroidal anti-inflammatory drug (NSAID) and frequently detected in aquatic environments, recovery studies explicitly evaluating the toxicity of this pharmaceutical are not very common. However, a few chemical characteristics of NPX could assist us in explaining the nature of the results observed in this study. Compared to IBU drugs of equal concentrations, the extended duration of pain relief after a single dose of naproxen has been widely established. Naproxen exhibits a biological half-life of approximately 15 hours, whereas ibuprofen has a half-life of around 2 hours (Haley & von Recum, 2019). It is worth noting that NPX also has a half-life of 14.6 days in aquatic environments (Wang et al., 2022). Due to its prolonged half-life and pharmacological properties, naproxen is commonly prescribed to address acute pain and inflammation associated with chronic conditions, including post-operative pain, migraines, gout, and rheumatoid arthritis (Stoev et al., 2021). In this scenario, the daphnids exposed to NPX and subsequently introduced to a pharmaceutical-free environment in our study may experience a prolonged recovery period because they might continue to endure the effects of NPX longer than when exposed to IBU.

Upon entering the aquatic environment, pharmaceuticals undergo various processes, including biodegradation and photodegradation, leading to the formation of new compounds (Śliwka-Kaszyńska et al., 2019). Photolysis emerges as a key mechanism for generating novel chemical compounds in water. This photodegradation process significantly impacts the persistence and toxicity of pollutants within the water (Śliwka-Kaszyńska et al., 2019) (Wojcieszyńska & Guzik, 2020). In the case of NPX, upon release into water bodies, it undergoes photodegradation, resulting in the detection of three degradation products in aquatic environments (Śliwka-Kaszyńska et al., 2019). Remarkably, these products exhibit higher persistence and potential toxicity than the primary compound of NPX, raising concerns about their potential accumulation in the environment (Cory et al., 2019). Hence, NPX may have been susceptible to photodegradation during the exposure phase, leading to the release of transformation products into the water. These transformation products exhibit increased toxicity, impacting our study's daphnids. Interestingly, the F4 generation of organisms derived from the NPX exposure period may still suffer from the lingering toxic effects of these transformation products, resulting in a slower recovery than the daphnids exposed to IBU. A few studies also observed the increase in toxicity of transformation products of NPX in aquatic organisms (Maculewicz et al., 2022) (Ma et al., 2014) (Cory et al., 2019) (Isidori et al., 2005), which corroborates the discussion of our findings in this study.

Allocation of energy is also a possible mechanism that the daphnids could have utilised to control the toxic effects of the NSAIDs. Organisms from the NPX single concentrations of the F4 generation produced fewer neonates than the daphnids allocated in the IBU treatments, and this could have happened due to the allocation of energy towards growth or detoxification processes rather than reproduction. This observation aligns with the findings of Heckmann et al. (2007), who reported a trade-off mechanism in Daphnia magna after exposure to NSAIDs. They observed an increase in the body size growth of organisms following exposure to IBU. Similarly, Alkimin et al. (2020) noted the activation of the detoxification system in Daphnia magna after exposure to ketoprofen. These studies provide further evidence supporting the effects on reproduction and highlight the complex responses of organisms to NSAID exposure.

The presence of pharmaceutical mixtures in aquatic environments is a growing global concern due to the challenge of removing them. Once they enter water, these chemical compounds undergo treatment in wastewater treatment plants. Among these drugs, NSAIDs are notably abundant in raw wastewater (Lolić et al., 2015; Adeleye et al., 2022; Rastogi et al., 2021). Specifically, ibuprofen and naproxen, among the NSAIDs class, are frequently detected in surface waters (Osman et al., 2024; Moreno Ríos et al., 2022).

Our study observed that reproduction, size, and behavioural endpoints were sensitive indicators of ibuprofen and naproxen toxicity when daphnids were exposed to environmental concentrations. The total number of neonates decreased at the end of 21 days in all generations compared to the control group, with males and ephippia consistently observed across generations. Despite exposure to a medium without NSAIDs, the daphnids did not recover after one generation (F4). However, the average number of neonates was higher in a few concentrations compared to the previous generation (F3). Additionally, regarding the binary mixture assessment, it can be concluded that the toxic effects of the mixtures are overall higher than the individual effects of each drug.

This study emphasises the importance of understanding pharmaceutical interactions and the implications of potential increased toxicity on aquatic ecosystems. Furthermore, it underscores the significance of evaluating whether the overall toxicity is caused by the mixture or led by only one component. We currently have limited data on specific compounds and their mixtures; therefore, further studies on pharmaceutical mixtures are needed to identify the most vulnerable species, comprehend how these interactions influence aquatic life, and improve risk assessment and management of pharmaceuticals and other chemical compounds entering water bodies.

Competing interests

The authors have no relevant financial or non-financial interests to disclose.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Author Contribution

Helena da Silva Viana de Souza (corresponding author): conceptualisation, data curation, formal analysis, investigation, methodology, writing- original draft, writing-review and editing. Dr. Anu Kumar: conceptualisation, methodology, supervision, writing- review and editing. Dr. Dayanthi Nugegoda: conceptualisation, supervision, writing- review and editing.

Acknowledgement

The authors thank the Royal Melbourne Institute of Technology for the RMIT Research Stipend Scholarship supporting Helena da Silva Viana de Souza.

Data Availability

Data is provided in the supplementary information section as IBU data, NPX data and Binary mix NSAID data.

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Multigenerational exposure of Daphnia carinata to NSAIDs in individual concentrations and binary mixtures

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Abstract

Figures

Introduction

Material and Methods

2.1. Pharmaceuticals

2.2. Cladocerans

2.3. Experimental design- multigenerational exposure

2.4. Reproduction recovery

2.5. Binary mixture toxicity assessment

3.0. Statistical analysis

Results

4.2. Multigenerational exposure of Daphnia carinata

4.2.1. Reproduction traits

4.2.2. Daphnia carinata body length

4.3. Behavioural effects

4.4. Post-exposure assessment

4.5. Binary mixture assessment on reproduction

Discussion

5.1. Survival and exposure conditions

5.2. Multigenerational assessment

5.2.1. Presence of males and ephippia

5.3. Toxic Units Mixture Assessment

5.4. Recovery assessment

Conclusions

Declarations

Competing interests

Funding

Author Contribution

Acknowledgement

Data Availability

References

Table 1

Additional Declarations

Supplementary Files

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