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Article

Low Concentrations of Ibuprofen Had No Adverse Effects on Deleatidium spp. Mayfly Nymphs: A 7-Day Experiment

by
Niña Sarah P. Batucan
1,*,
Louis A. Tremblay
2,3,4,
Grant L. Northcott
5 and
Christoph D. Matthaei
1
1
Department of Zoology, 340 Great King Street, Dunedin 9016, New Zealand
2
Cawthron Institute, Nelson 7010, New Zealand
3
School of Biological Sciences, University of Auckland, Auckland 1142, New Zealand
4
Manaaki Whenua—Landcare Research, Lincoln 7608, New Zealand
5
Northcott Research Consultants Limited, Hamilton 3200, New Zealand
*
Author to whom correspondence should be addressed.
Environments 2025, 12(4), 102; https://doi.org/10.3390/environments12040102
Submission received: 26 February 2025 / Revised: 18 March 2025 / Accepted: 25 March 2025 / Published: 27 March 2025
(This article belongs to the Special Issue Environmental Risk Assessment of Aquatic Environments)
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Abstract

:
Concerns over pharmaceutical contaminants are increasing due to their high biological activity and ubiquity, with wastewater being the main source. Ibuprofen is extensively used worldwide and commonly detected in freshwaters due to its low degradability during wastewater treatment. Daphnia magna is the most-used model taxon for toxicity testing of ibuprofen, but this crustacean is known to be less sensitive to some contaminants than several freshwater insect groups. Our study assessed the toxicity of ibuprofen (nominal concentration range 2.0–2147.5 µg/L) to a native New Zealand mayfly, Deleatidium spp., in a 7-day static renewal experiment, with the neonicotinoid imidacloprid (1.4 µg/L) as a reference toxicant. Test concentrations of ibuprofen included three field-realistic and four higher concentrations that might occur in severely polluted streams. Mayfly responses indicated some negative trends (decreased survival and moulting propensity, increased impairment and immobility), but all patterns were non-significant. The imidacloprid control also had no significant impacts but tended to increase mayfly impairment. Overall, Deleatidium nymphs were largely unaffected by the entire range of experimental ibuprofen concentrations, suggesting that ibuprofen may be a relatively benign stressor for these organisms, although longer-term exposure experiments are needed to confirm if they demonstrate susceptibility to chronic exposure.

Graphical Abstract

1. Introduction

Pharmaceutical contamination is a global issue affecting freshwater systems [1]. Concerns regarding their environmental hazard are increasingly salient, even though these compounds are typically detected at the ng–μg/L range, as they are highly biologically active [2,3]. Pharmaceutical compounds mainly reach freshwater systems through being discharged from wastewater treatment facilities, which do not effectively remove many contaminants of emerging concern [1,3,4].
Ibuprofen is among the most-detected pharmaceutical contaminants worldwide, owing to its widespread use and accessibility over the counter [5]. Ibuprofen is a non-steroidal anti-inflammatory drug (NSAID) used to treat pain and inflammation in both humans and animals [6]. Due to its physicochemical properties, ibuprofen is not readily degraded in the environment [7]. Because of this, and due to its ongoing extensive use, ibuprofen is continually replenished in recipient freshwater environments, thus highlighting its potential risk to aquatic life [8].
The crustacean Daphnia magna is currently the most-used model taxon in testing the toxicity of NSAIDs [9]. The findings from this model taxon may not necessarily be protective of crustaceans from other ecosystems globally. Additionally, crustaceans may not be the most sensitive taxa to contaminants. For instance, a review by Morrissey et al. [10] found that Crustacea are 1000 times less sensitive to neonicotinoid insecticides than some other invertebrates, particularly those belonging to the orders Ephemeroptera, Trichoptera and Diptera. Consequently, ecotoxicological studies involving ibuprofen or other NSAIDs on EPT (Ephemeroptera, Plechoptera, Trichoptera) taxa may allow developing a more comprehensive picture of the environmental hazards of these drugs and other classes of contaminants.
Ephemeroptera are among the most sensitive taxa to environmental perturbations [11]. Larvae of the mayfly Deleatidium spp. are among the most common and ubiquitous invertebrates in New Zealand streams and rivers [12] and are sensitive to a wide range of pollutants. Deleatidium spp. belongs to the family Leptophlebiidae, which is one of the largest families of mayflies worldwide and comprises the highest number of genera [13].
The aim of this study was to characterise the toxicity of environmentally relevant concentrations of ibuprofen to Deleatidium mayfly nymphs. We hypothesised that (H1) increasing ibuprofen concentrations will lead to reduced mayfly survival rates and moulting propensity (used as a proxy for larval growth), as contaminant exposure tends to negatively affect their metabolism and general homeostatic processes [14,15]; and (H2) increasing doses of ibuprofen will lead to higher proportions of impaired and immobile nymphs, as general devitalisation of an organism can manifest behaviourally [9,16], and impairment and immobility are well-established stress responses in Ephemeroptera [17,18]. Because the mechanism of toxicity of ibuprofen in Deleatidium remains undefined, we used the neonicotinoid imidacloprid as a reference toxicant, as the detrimental effects of the latter on Deleatidium spp. are well established [18,19].

2. Materials and Methods

2.1. Chemicals, Water Media and Mayfly Food

Analytical-standard grade rac-ibuprofen (98% purity, CAS no. 15687-27-1) was purchased from Toronto Research Chemicals (PM Separations, Queensland, Australia), and imidacloprid (PESTANAL®, CAS no. 138261-41-3) from Sigma-Aldrich (Sigma-Aldrich, Castlehill, NSW, Australia). Anhydrous dimethyl sulfoxide (DMSO) was acquired from Sigma-Aldrich (Merck, Auckland, New Zealand) and HPLC grade dichloromethane from ThermoFisher (ThermoFisher Scientific, Auckland, New Zealand). We used artificial stream water (ASTW), following the formula developed by the American Society for Testing Materials, which consisted of double-distilled water (Gemini-MB Ultra High Purity Water System; Aries FilterWorks, West Berlin, NJ, USA), and added 0.57 NaHCO3, 0.17 CaSO4·2H2O, 0.25 MgSO4·7H2O and 0.03 KCl in mM/L. ASTW was prepared 48 h before the experiment, stored in 25 L polyethylene jerrycans, allowed to normalise to the climate-controlled room temperature, and aerated 24 h before the experiment commenced. Water media parameters were measured in the jerrycans containing the ASTW on Days 0 and 6 of the experiment using YSI Pro2030 (Yellow Springs, OH, USA), with the following results: dissolved oxygen 95.75% (±2.36 SD [standard deviation]), water temperature 10 °C (±0.14 SD), and salinity 0.1 (±0 SD). Using pHep®4 (Hanna Instruments, Woonsocket, RI, USA), pH was measured at 7.17 (±0.52 SD).
Concentrated stock solutions of ibuprofen (45 mg/mL in DMSO) and imidacloprid (10 mg/mL in acetone) were spiked separately into ASTW to produce working standards, using a magnetic stirrer for mixing. Working standards of 225 and 112.5 μg/L ibuprofen were used to spike 2.5 L and 1.5 L working stock solutions for the 48 h and 6 d media replenishment, respectively (further details in Section 2.5). A working standard of 10 μg/L imidacloprid was used for both replenishment modalities.
Ceramic tiles (10 × 10 × 1.8 cm) were used as substratum for Deleatidium nymphs during the acclimatisation and experimental periods, and as a substratum for periphyton, which was the food source for the nymphs. These tiles were placed at a site in Lindsay Creek, located in North East Valley, Dunedin, New Zealand (45.8420° S, 170.5408° E) in June 2022, to allow for a 2 w periphyton colonisation prior to the start of the experiment. The stream was selected as it drains a nature reserve clad in native forest and has a flow regime that rarely causes marked bed disturbance [18]. The stream is easy to access and has natural presence of Deleatidium. Further, a previous study by Bernot et al. [20] at this site found no detectable residues of ibuprofen above the method detection limit.

2.2. Mayfly Collection

Deleatidium specimens were wild-caught in a 75 m reach of Silver Stream, a fourth-order stream located in a native forest catchment north of Dunedin, New Zealand (45.8096° S, 170.4211° E) on 29 June 2021. A pulsed-DC backpack electro-shocker (Kainga EFM300; NIWA, Christchurch, New Zealand) and a rectangular pole net (0.9 × 0.7 m, mesh size 3 mm) were used to catch drifting insects for efficiency and to minimise damage on the nymphs see [18,21]. The invertebrates were transferred from the net to sorting bins for the selection of early-instar nymphs measuring 5–8 mm (head-to-body length). This size range has been demonstrated to survive better under laboratory conditions and less likely to emerge during chronic experiments [18].
To maximise their survival, mayfly nymphs were kept in buckets with aerated stream water (using battery-powered pumps—Aqua One Battery Air 250; Aqua One, Ingleburn, NSW, Australia) during the transit between the sampling site and laboratory. Once in the climate-controlled rooms, the nymphs were allowed to acclimatise in the buckets overnight. The following day, they were transferred to 40 L holding bins containing 20 L of aerated ASTW and pre-colonised algal tiles, where they were allowed to acclimatise for a further 48 h. After that period, nymphs with intact legs and cerci were randomly assigned to the experimental glass chambers containing the treatment media and algal tiles up to n = 20. This transfer marked the beginning of the experiment, on 1 July 2022. The nominal temperature in the climate-controlled rooms was set at 9 °C, as a previous related experiment showed this temperature resulted in the best survival of Deleatidium nymphs [21]. The measured room temperature was 10.80 ± 0.73 °C for the duration of the experiment. A 12:12 light:dark regime with a 1 h ramp between light–dark phases was applied. Laboratory conditions were the same during acclimatisation and experimentation.

2.3. Experimental Design

A 7-day static-renewal test was conducted with seven nominal concentrations of ibuprofen: 2.0, 6.4, 20.5, 65.5, 209.7, 671.1, and 2147.5 µg/L. These had a scaling factor of 3.2, as recommended by Moermond et al. [22]. The concentration range for ibuprofen encompassed all environmentally relevant concentrations, as the highest concentration measured to date in running waters worldwide is 32 μg/L [23]. We extended this concentration range by another two magnitudes to allow testing for effects at higher concentrations, which might occur in severely polluted urban streams. Three controls were used: blank , solvent (DMSO 0.004% v/v), and positive (imidacloprid, nominal concentration 1.4 µg/L). Imidacloprid was used as a reference toxicant as its effects on Deleatidium nymphs have been well characterised, e.g., [18].
We used 1 L glass treatment chambers (internal dimensions 11.6 × 16 × 5.7 cm) with plastic clip-on lids containing 500 mL of treatment solution. The experimental design consisted of five replicates per treatment (7× IBF concentrations and 3× blank controls). The chambers were continuously aerated using electric air pumps (Aqua One Precision 9500 Airpump; Aqua One, Ingleburn, NSW, Australia) via a 1 mL plastic pipette tip inserted through the top of the lids to provide aeration into the surface of the treatment solutions. Algal tiles and ASTW were replaced every 6 d; in between, the treatment and blank media solutions were replenished every 48 h. Replenishment involved syphoning out 80% of test ASTW solution and replacing it with an equal volume of fresh solution to maintain ibuprofen concentrations. An ibuprofen chemical fate pilot study showed reductions of up to 46% over seven days under similar laboratory conditions in the absence of mayfly nymphs (Figure S1). This reduction amounts to a daily loss of approximately 7%. The 48-h replenishment with fresh treatment solutions was considered the best compromise between maintaining ibuprofen concentrations and minimising disturbance to the mayfly nymphs. The chambers were checked daily and any exuviae removed.

2.4. Mayfly Endpoints

Mayfly survival and moulting propensity were recorded as the percentage of nymphs alive or exuviae, respectively, over the seven days. Nymph impairment and immobility were recorded (also as percentages) as further sublethal responses on Day 7. Following Hunn et al. [18], impairment was defined as the inability of the nymph to swim or right itself when placed ventral side up, whereas immobility was when the nymph was unable to move besides the twitching of appendages.

2.5. Water Chemical Analysis

Three randomly selected ibuprofen and imidacloprid treatment replicates from each set of five were sampled at Day 0 and Day 6 of the experiment to quantify potential changes in concentrations over time. For ibuprofen, only low (2.0 μg/L), medium (65.5 μg/L) and high (2147.5 μg/L) concentrations were analysed due to resource restrictions. The blank and solvent samples were not sampled as no toxicants were added to these. To minimise cross-contamination, glass treatment chambers containing the blank and solvent controls (in this order) were prepared before those containing the ibuprofen treatment. Aliquots of 10 mL were sampled from the glass chambers by suctioning using a 10 mL pipette and transferred into precleaned 15 mL amber glass EPA vials. Two millilitres of dichloromethane (DCM) was added to the vials, secured with the Teflon-lined caps, and the contents were vortexed for 30 s to capture and preserve the dissolved ibuprofen from ASTW and inhibit microbial activity. No DCM was added to the imidacloprid samples as they were shown to be stable for 2 years after sampling (see Table 1). All samples were stored at 4 °C in the dark until analysis.
The ibuprofen samples were extracted in three batches corresponding to the three assessed treatment concentrations. Each batch of treatment solutions included quality assurance (QA) samples comprising an extraction water blank (10 mL of purified Milli-Q lab water) and an equivalent Milli-Q lab water blank spiked with ibuprofen and its metabolites 1-hydroxyibuprofen, 2-hydroxyibuprofen, and carboxyibuprofen. A mixed surrogate standard containing the surrogate recovery compounds MCPB, naphthalene acetic acid (NAA) and dichlorprop were added to all treatment and QA samples to assess the efficacy of the extraction and analysis method for each treatment and QA sample. The concentrations of ibuprofen and the three metabolites (QA spike samples) spiked into the treatment solutions were comparable to the nominal concentrations of ibuprofen in the three treatments (2.0, 50 and 200 μg/L). Similarly, the concentrations of surrogate recovery compounds spiked into the treatment and QA samples were comparable to the nominal concentrations of ibuprofen in the three treatments. The pH levels of the treatment and QA sample solutions were adjusted to pH 2 by adding 0.01 mL of 6N HCl to the 10 mL volume.
The ibuprofen sample vials were vortexed, and the previously added DCM was allowed to separate and settle at the bottom of the vials. The lower DCM layer was removed with a glass Pasteur pipette and transferred into a tapered test tube. The aqueous solution in the original vial was extracted two more times with 2 mL of DCM, with the DCM extracts being combined in the test tube. DCM extracts were then concentrated under a stream of nitrogen to a volume of 2 mL. Residual moisture was removed from the DCM extracts by passing the extracts through a mini-column of anhydrous sodium sulphate (Na2SO4). The DCM extracts from the low treatment solutions were collected into a 5 mL glass conical-bottom Reacti-Vial™ along with three 1 mL DCM rinses of the original tapered test tube. The dried DCM extracts were fully evaporated under a gentle stream of nitrogen gas, reconstituted in 0.25 mL of acetone and transferred into a 1 mL Reacti-Vial™. The 5 mL Reacti-Vial™ was rinsed with another three times with 0.25 mL of acetone, with each rinse transferred into and combined within the 1 mL Reacti-Vial™.
The column-dried extracts from the medium and high treatment solutions were collected in 5 and 10 mL volumetric flasks, respectively, and adjusted to 5 or 10 mL with DCM as an initial dilution step. A 1 mL aliquot from the medium concentration treatment DCM extracts (taken from 5 mL) and a 0.1 mL aliquot from the high treatment DCM extracts (taken from 10 mL) were transferred into individual 1 mL glass Reacti-VialsTM (second dilution step).
The dilution of the DCM extracts from the medium and high treatments could compromise the detection of any ibuprofen metabolites produced during the 6 d exposure period. Therefore, the remaining 4 mL of DCM extracts from the medium treatment solutions were evaporated to dryness and transferred with acetone washes into 1 mL glass Reacti-VialsTM. Lastly, 1.5 mL aliquots of the DCM extracts from the high treatment solutions were transferred into 2 mL glass Reacti-VialsTM.
In preparation for derivatisation, an internal standard solution containing ibuprofen-d3, monobutylphtlate-d4 and monoethylhexylphthalate-d4 was added to the sample extracts in 1 mL Reacti-VialsTM. These extracts were evaporated to dryness under a stream of nitrogen gas. Then, 0.50 µL of ethyl acetate and 50 µL of BSTFA with 1% TMCS derivatisation reagent (Merck, Auckland, New Zealand) were added to the vials, which were capped, vortexed to mix, and incubated at 70 °C for 20 min. Following incubation, the contents of the Reacti-VialsTM were vortexed again and returned to room temperature, 0.4 mL of ethyl acetate added, then vortexed again. The derivatised sample extracts were transferred into gas chromatography (GC) vials with Teflon micro-insert septa and refrigerated until analysis by GC-MS (described in Supplementary Information).
To prepare the imidacloprid samples for analysis, 0.2 mL of the samples were transferred into a GC vial followed by 0.2 mL of 0.1% acetic acid in acetonitrile and 0.6 mL acetic acid ammonium formate buffer at pH 4. These GC vials were stored under refrigeration until analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS, described in Supplementary Information).

2.6. Statistical Analysis

All statistical analyses were conducted using the statistical software R (version 2023.03.0). Significance was set at α = 0.05. Separate log–log binomial generalised linear modelling (GLM) was performed for survival, moulting propensity, impairment, and immobility using the stats package. The blank and solvent control treatments were pooled as no significant differences were detected between the two [22,24]. Overdispersion was checked using the function code by Bolker [25], and quasibinomial distribution was used to correct overdispersed data. McFadden’s pseudo-R2 (p2) was calculated to obtain standardised effect sizes. Values between 0.2 and 0.4 represent an excellent model fit [26]. In our study, we used a conservative approach that considered p2 values >0.4 to be a strong effect and <0.2 a weak effect.
The effects induced by the highest concentration of ibuprofen (2147.5 µg/L, IBF-HIGH) were compared with the control and the imidacloprid control (IMIDA, 1.4 μg/L). As in the GLMs above, blank and solvent controls were pooled in the no-toxicant control. Analysis of variance (ANOVA, stats package) was performed to evaluate statistical differences in survival, moulting, impairment, and immobility across the three groups. Here, the effects package was used to calculate eta squared (η2) as standardised effect sizes, where ≤0.10 = small, ≤0.30 = medium, and ≤0.50 = large [27]. Levene’s tests (car package) were used to check for homoscedasticity. Where a significant overall difference was detected in the ANOVA, pairwise post hoc Tukey’s tests (stats package) were performed to detect which groups differed from each other.

3. Results

3.1. Measured Ibuprofen and Imidacloprid Concentrations

The median measured concentrations of low (2.0 μg/L), medium (65.5 μg/L) and high (2147.5 μg/L) concentrations of ibuprofen are presented in Table 1. The mean concentration of ibuprofen measured in the medium and high treatment solutions at Day 0 and Day 6 were statistically indistinguishable, as indicated by the overlap in their confidence intervals. In contrast, the concentration of ibuprofen measured in the low treatment solution decreased by 31% from Day 0 to Day 6, downward-skewing the median between the two days to such an extent that it fell outside the acceptable margin of reliability of 20% of nominal, as in Moermond et al. [22]. The median concentrations between Days 0 and 6 for the medium and high ibuprofen treatments were within the acceptable margin of reliability [22]. Analyses of the QA samples and accompanying discussion on the possible fate of ibuprofen loss from the water medium are found in the Supplementary Information. Given that the median recovered lowest treatment concentration was not substantially lower (i.e., not more than 50% loss) than nominal and the median recovered medium and high treatment concentrations were within the reliable margin, it is likely that when these values are extrapolated to the in-between concentrations they were close to the nominal concentrations. Even if the measured ibuprofen concentrations were lower (or slightly higher) than target concentrations, they would still remain within environmentally relevant concentrations. Moreover, they did not elicit any obvious toxicity responses in our model taxon (explored further in Section 3.2). Finally, as the blank and solvent controls were not analysed for the presence of ibuprofen, we cannot entirely rule out the possibility of contamination. However, because no ibuprofen was added to these controls in our highly controlled experiment and our achieved ibuprofen concentrations were generally robust, it is extremely unlikely that such contamination occurred to an extent that it affected our conclusions regarding the effects of the ibuprofen addition treatments. Thus, notwithstanding the above limitations and that we have provided further analyses to account for the fate of lost concentration as per [22], our study has maintained reasonable methodological rigour; therefore, we proceed with presenting our remaining results.
The median achieved imidacloprid concentration (1.77 μg/L) was outside the margin of reliability (20% of nominal) [22]. However, as the imidacloprid treatment was our positive control, keeping within the 20% margin was not crucial and it still served its function as the reference toxicant. The median concentrations of ibuprofen and imidacloprid from the triplicate samples taken on Days 0 and 6 are used in subsequent reporting.

3.2. Mayfly Responses to Ibuprofen Dose Concentrations

Increasing ibuprofen concentrations caused a decline in mayfly nymph survival that was borderline significant, with a weak effect size (Table 2; Figure 1a). Moulting propensity declined very slightly, but this pattern was clearly not significant (Table 2; Figure 1b). Impairment and immobility both increased marginally (Figure 1c,d; see slopes in Table 2), with borderline significant p-values and weak effect sizes.

3.3. Comparisons with No-Toxicant and Imidacloprid Controls

There was a borderline significant difference in survival between control, IMIDA, and IBF-HIGH (Table 3), where survival tended to be lowest in IBF-HIGH (Figure 2a). For moulting propensity, a significant overall difference was detected across the three treatments (Table 3). Post hoc analysis showed that moulting in IBF-HIGH was significantly lower than in IMIDA but was comparable to the control (Figure 2b). Mayfly impairment tended to be highest in IMIDA (Figure 2c), but this pattern was not significantly different to the other treatments (Table 3). Finally, there were generally very few immobile mayfly nymphs (<0.5%) (Figure 1d and Figure 2d), and no significant difference was detected between treatments (Table 3).

4. Discussion

Our study examined the toxicity of seven concentrations of ibuprofen (1.45–2070 µg/L) using Deleatidium mayfly nymphs as model taxon. The three lower concentrations were field-realistic based on data from existing freshwater surveys across the world [23], whereas the four higher concentrations might occur in severely polluted streams. We found that, within our concentration range and 7 d exposure duration, increasing doses of ibuprofen had no adverse effects on Deleatidium based on the endpoints assessed. Compared to imidacloprid, the highest concentration of ibuprofen caused significantly lower moulting propensity, but both were no different to the control. These findings did not support our hypotheses predicting negative effects on the determined endpoints of nymph survival and moulting propensity (H1) or impairment and immobility (H2).
In previous related work, the LC5014d (14 d median lethal concentration) of ibuprofen for the crustacean Daphnia magna was 80 mg/L [28]. A study by Han et al. [29] reported that the NOEC21d (the 21 d No Observed Effect Concentration) on survival for D. magna was 33.3 mg/L. They also found a 20–30% reduction in survival in the crustacean Moina macrocopa after 21d exposure to up to 50 mg/L of ibuprofen, but this decline was not significant. In another study with D. magna [30], the LC5072h (72 h median lethal concentration) of ibuprofen was 8.33 mg/L, while the LC5021d was 3.97 mg/L. A study on the shrimp Neocaridina denticulata established an LC5096h of 6.60 mg/L [31]. Other studies on non-arthropod models also found low sensitivity to ibuprofen. For example, the gastropod Planorbis carinatus had an LC5072h of 17.1 mg/L and a NOEC21d of 5.36 mg/L [32], while the cnidarian Hydra attenuata had an LC5096h of 22.36 mg/L [33]. The above concentrations were all notably higher than those from our study, and their exposure durations varied, with some being three times longer than our 7 d experiment. Nevertheless, all these related studies parallel our finding of no significant mortality in our mayfly model taxon after exposure to up to 2.1 mg/L ibuprofen for 7 d. By contrast, Muñiz-González [34] reported that larvae of the midge Chironomus riparius were highly sensitive to ibuprofen, with survival rates that significantly declined by 25% after exposure to 1 μg/L ibuprofen for just 96 h. This striking difference highlights the variability of toxicant sensitivities across certain taxa and emphasises the need to use model species across a range of taxonomic groups in environmental risk assessment.
Interestingly, the imidacloprid treatment also did not reduce Deleatidium nymph survival in our experiment. Even for imidacloprid, 7 d may be too short a duration to capture significant mortality effects on this model taxon at a concentration of 1.77 μg/L, although some such effects started to appear in Deleatidium nymphs after 7 d at similarly low imidacloprid concentrations during 28 d exposure to 0.05–4.24 μg/L of this insecticide [35].
Moulting propensity of Deleatidium nymphs did not decline significantly with rising ibuprofen concentrations, suggesting that the growth of the nymphs was not affected. In related research, ibuprofen at 20–80 mg/L (14 d exposure) caused a significant increase in the body surface area of Dapnia magna [36], whereas a significant decline in the intrinsic growth rate of the same species occurred after 21 d exposure to 50 μg/L of ibuprofen [35]. Further, Pounds et al. [31] reported that the snail Planorbis carinatus showed significantly reduced growth after 14–21 d of exposure to 3.2 mg/L of ibuprofen. Based on these related findings, ibuprofen might cause chronic effects on Deleatidium nymph growth during exposure periods exceeding 7 d.
Deleatidium nymph moulting propensity also did not differ between the imidacloprid and control treatments, likely due to the short duration of exposure, as discussed earlier. This interpretation is supported by the findings of the abovementioned 28 d exposure experiment by Macaulay et al. [18] focusing on imidacloprid (0.05–4.24 μg/L) effects on Deleatidium, where a decline in nymph moulting propensity occurred during Days 8–14 and 15–21, but not during Days 0–7.
Both mayfly impairment and immobility tended to increase weakly with rising ibuprofen concentrations in our 7 d study, with borderline significant p-values. Previous related short-term studies on locomotion of the crustacean Gammarus pulex [36] reported a biphasic response to ibuprofen exposures of 2 h, with decreased locomotion at lower concentrations (1–100 ng/L) but increased locomotion at higher concentrations (1–1000 μg/L). In another short-term study [37], a 20 min exposure to environmentally relevant concentrations of ibuprofen (0.21 μg/L) caused a chemorepellent effect on the ciliate Tetrahymena pyriformis, where exposure caused ciliates to move away from the dose source. Two further short-term studies examined locomotive effects in fish. Xia et al. [38] reported that newly hatched zebrafish Danio rerio showed decreased spontaneous movement and swimming performance (50–500 μg/L for 2 h), while Ogueji et al. [39] found that the catfish Clarias gariepinus showed a loss in swimming coordination (280–480 μg/L for 96 h). Overall, the existing evidence on locomotion effects of ibuprofen is conflicting, with both hyper- and hypoactive effects occurring at various concentrations, exposure durations and in different model taxa. To our knowledge, no studies have investigated invertebrate locomotive behavioural responses to long-term exposure (>15 d; [39]) to environmentally relevant ibuprofen concentrations, and the same can be said for vertebrates.
Our results showed a weak, marginally significant trend for hypoactive effects (i.e., more impairment or immobility) of ibuprofen on Deleatidium nymphs. This somewhat inconclusive result may be due to our relative short exposure duration of 7 d, and/or that ibuprofen may not exert a strong neuroactive mode of action on Deleatidium. Indeed, even for imidacloprid, which was designed to be neurotoxic to insects, no significant effects on mayfly impairment or immobility were found in our study. Our achieved imidacloprid concentration (1.77 μg/L) was slightly higher than the 7 d EC50 (1.21 μg/L) for the impairment of Deleatidium in [18]. Although our 7 d experiment resulted in no statistically significant impairment in mayfly nymphs, the response pattern trends (higher percentage of impaired mayflies in the imidacloprid treatment than in the control and the highest ibuprofen treatment) aligned with our expectations, and a longer exposure could have made the imidacloprid effect more evident. Immobility, a more severe response to a toxicant, was rare in all our treatments. In Macaulay et al.’s [18] study, the EC507d for immobility was 2.60 μg/L, reducing to 1.09 μg/L by 11 d in Deleatidium nymphs. Thus, the rarity of immobile mayflies in our study is likely attributable to the short exposure time.

5. Conclusions

The range of ibuprofen concentrations investigated in our 7 d study showed negligible toxicity to Deleatidium mayfly nymphs, although the response directions in all four studied mayfly endpoints (survival, moulting, impairment, immobility) trended towards what we had hypothesised as toxic responses. It is possible that longer exposure times would have revealed clearer response patterns. The short duration of our study is a limitation and highlights the need for longer-term exposures which are still rare in ecotoxicological research on pharmaceuticals [40], particularly when testing environmentally realistic concentrations. Further, tests involving molecular biomarkers might have provided a better understanding of the toxicity effects in our study, especially given the short exposure duration; thus, future related studies may benefit from using such suborganism-level markers besides the endpoints we studied [41,42]. Decisions for environmental policy are generally based on a weight-of-evidence approach [43,44]. Consequently, there is an urgent requirement for increased environmental realism within the heterogenous body of evidence on the ecotoxicological effects of pharmaceuticals and other emerging contaminants, besides the prevailing goal of establishing dose-dependent, cause-and-effect relationships between contaminants and biological endpoints [45,46].
Moreover, contaminants are mainly found in mixtures in the natural environment, and they interact not just with each other, but also with abiotic factors that are dynamically changing in the environment [45]. For example, greater growth inhibition on the alga Chlorella vulgaris was observed with ibuprofen mixed with the antibiotic ciprofloxacin than with ibuprofen alone [47]. Indeed, there is a paucity of evidence on the effects of contaminant mixtures at environmentally relevant concentrations, particularly from field-realistic experimental studies [48]. Future studies will need to bridge this gap so that the effects of a myriad of emerging and other contaminants co-occurring in the freshwater environment can be better understood and aligned with the principles of One Health [49].

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/environments12040102/s1, Figure S1 Results from the ibuprofen recovery pilot study; Analysis of the concentration of ibuprofen in experimental treatment solutions; Table S1. Retention times and quantifier/qualifier ions used for the detection of trimethylsilyl (TMS) derivatives of internal standard, target analyte (ibuprofen and metabolites) and surrogate standard compounds; Analysis of the concentration of imidacloprid in experimental treatment solutions; Table S2. Mean recovery ± 95% confidence interval of surrogate compounds spiked into all extracted treatment samples and associated Quality Assurance samples; Table S3. Recovery of ibuprofen and its metabolites spiked into all extracted treatment samples and associated Quality Assurance samples.

Author Contributions

Conceptualisation, N.S.P.B. and C.D.M.; methodology, N.S.P.B., G.L.N. and C.D.M.; formal analysis, N.S.P.B.; investigation, N.S.P.B.; resources, C.D.M., L.A.T. and G.L.N.; data curation, N.S.P.B.; writing—original draft preparation, N.S.P.B.; writing—review and editing, N.S.P.B., L.A.T., G.L.N. and C.D.M.; visualisation, N.S.P.B.; supervision, L.A.T., G.L.N. and C.D.M.; project administration, N.S.P.B.; funding acquisition, L.A.T., G.L.N. and C.D.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was partly funded by the Department of Zoology of the University of Otago, a New Zealand Ministry of Business, Innovation and Employment grant (CAWX1708), and by Strategic Science Investment Funding (SSIF) for Crown Research Institutes from the New Zealand Ministry of Business, Innovation and Employment (PRJ4655).

Data Availability Statement

The data, R code, and spreadsheet containing the measured concentrations of individual water samples can be accessed through this link, https://figshare.com/s/fce849c194f0e50e7730 accessed on 18 March 2025.

Acknowledgments

We would like to thank Daniel Zamorano, Nayla Rhein, Nat Lim, and the many student volunteers from the Department of Zoology of the University of Otago for their incredible help in the intensive work of capturing and sorting the Deleatidium mayfly nymphs needed for the experiment.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Environments 12 00102 g001
Figure 1. Log–log binomial GLMs of Deleatidium mayfly nymph endpoints in response to increasing ibuprofen exposure, (a) Survival; (b) Moulting propensity; (c) Impairment; (d) Immobility. Red lines indicate means, dashed lines indicate 95% CIs, and error bars indicate SEs.
Figure 1. Log–log binomial GLMs of Deleatidium mayfly nymph endpoints in response to increasing ibuprofen exposure, (a) Survival; (b) Moulting propensity; (c) Impairment; (d) Immobility. Red lines indicate means, dashed lines indicate 95% CIs, and error bars indicate SEs.
Environments 12 00102 g001
Environments 12 00102 g002
Figure 2. Comparisons of mean percentages (using one-way ANOVAs) in the highest ibuprofen treatment (IBF-HIGH, n = 5) to the toxicant-free control (n = 10) and the reference toxicant (IMIDA, imidacloprid, n = 5) for all four mayfly endpoints: (a) Survival; (b) Moulting propensity; (c) Impairment; (d) Immobility. Error bars indicate SEs. Letters above the bars indicate post hoc rankings for moulting propensity.
Figure 2. Comparisons of mean percentages (using one-way ANOVAs) in the highest ibuprofen treatment (IBF-HIGH, n = 5) to the toxicant-free control (n = 10) and the reference toxicant (IMIDA, imidacloprid, n = 5) for all four mayfly endpoints: (a) Survival; (b) Moulting propensity; (c) Impairment; (d) Immobility. Error bars indicate SEs. Letters above the bars indicate post hoc rankings for moulting propensity.
Environments 12 00102 g002
Table 1. Mean achieved concentrations (±95% CI, Student t-test) of low, medium, and high concentrations of ibuprofen and imidacloprid on Day 0 and Day 6, with triplicate samples per concentration for each sampling event. Water samples analysed by gas chromatography. %N = percent of nominal; Median = median of pooled achieved start and end concentrations from Day 0 and 6. All concentrations are in µg/L.
Table 1. Mean achieved concentrations (±95% CI, Student t-test) of low, medium, and high concentrations of ibuprofen and imidacloprid on Day 0 and Day 6, with triplicate samples per concentration for each sampling event. Water samples analysed by gas chromatography. %N = percent of nominal; Median = median of pooled achieved start and end concentrations from Day 0 and 6. All concentrations are in µg/L.
Nominal Achieved
Start
Day 0
(n = 3)		%NAchieved
End
Day 6
(n = 3)		%NMedian
(n = 6)		%N
Ibuprofen
2.0 (Low)1.7 (±0.1)851.17 (±0.05)591.4573.0
65.5 (Medium)57.2 (±3)8757.5 (±7)8857.888.0
2147.5 (High, IBF-HIGH)2034 (±62)952090 (±43)97207096.4
Imidacloprid
1.4 (IMIDA)1.50 (±0)1072.03 (±0.29)1451.77126.4
Table 2. Summary of statistical outputs from log–log binomial (or quasibinomial) GLMs. No significant p-values were found in these analyses. df = degrees of freedom, p2 = McFadden’s pseudo-R2.
Table 2. Summary of statistical outputs from log–log binomial (or quasibinomial) GLMs. No significant p-values were found in these analyses. df = degrees of freedom, p2 = McFadden’s pseudo-R2.
dfsSlopeaz-/bt-Valuep-ValueEffect Size p2
Survival43−0.10−1.92 a0.060.076
Moulting43−0.03−1.27 b0.200.030
Impairment430.231.81 a0.060.095
Immobility430.291.71 a0.060.139
Table 3. Summary of statistical outputs from one-way ANOVAs comparing control (CONT, n = 10), imidacloprid at 1.77 µg/L as the positive control (IM, n = 5) and the highest concentration of ibuprofen exposure at 2147.5 μg/L (IBF-H, n = 5). Significant p-values are in bold. df = degrees of freedom; η2 = eta squared.
Table 3. Summary of statistical outputs from one-way ANOVAs comparing control (CONT, n = 10), imidacloprid at 1.77 µg/L as the positive control (IM, n = 5) and the highest concentration of ibuprofen exposure at 2147.5 μg/L (IBF-H, n = 5). Significant p-values are in bold. df = degrees of freedom; η2 = eta squared.
dfF-/Χ2-Valuep-Value η2Post Hoc Test Ranking
Survival173.440.060.24
Moulting173.660.0470.30CONT = IM, CONT = IBF-H, IM > IBF-H
Impairment170.910.420.10
Immobility170.620.620.06
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MDPI and ACS Style

Batucan, N.S.P.; Tremblay, L.A.; Northcott, G.L.; Matthaei, C.D. Low Concentrations of Ibuprofen Had No Adverse Effects on Deleatidium spp. Mayfly Nymphs: A 7-Day Experiment. Environments 2025, 12, 102. https://doi.org/10.3390/environments12040102

AMA Style

Batucan NSP, Tremblay LA, Northcott GL, Matthaei CD. Low Concentrations of Ibuprofen Had No Adverse Effects on Deleatidium spp. Mayfly Nymphs: A 7-Day Experiment. Environments. 2025; 12(4):102. https://doi.org/10.3390/environments12040102

Chicago/Turabian Style

Batucan, Niña Sarah P., Louis A. Tremblay, Grant L. Northcott, and Christoph D. Matthaei. 2025. "Low Concentrations of Ibuprofen Had No Adverse Effects on Deleatidium spp. Mayfly Nymphs: A 7-Day Experiment" Environments 12, no. 4: 102. https://doi.org/10.3390/environments12040102

APA Style

Batucan, N. S. P., Tremblay, L. A., Northcott, G. L., & Matthaei, C. D. (2025). Low Concentrations of Ibuprofen Had No Adverse Effects on Deleatidium spp. Mayfly Nymphs: A 7-Day Experiment. Environments, 12(4), 102. https://doi.org/10.3390/environments12040102

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