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Article

Anxiety in Duckweed–Metabolism and Effect of Diazepam on Lemna minor

by
Adéla Lamaczová
1,2,
Tomáš Malina
2,
Eliška Maršálková
2,
Klára Odehnalová
2,
Radka Opatřilová
1,
Petra Přibilová
2,
Štěpán Zezulka
2 and
Blahoslav Maršálek
2,*
1
Faculty of Pharmacy, Masaryk University, Žerotínovo nám. 1, 602 00 Brno, Czech Republic
2
Institute of Botany, Czech Academy of Sciences, Lidická 25/27, 602 00 Brno, Czech Republic
*
Author to whom correspondence should be addressed.
Water 2022, 14(9), 1484; https://doi.org/10.3390/w14091484
Submission received: 5 April 2022 / Revised: 27 April 2022 / Accepted: 3 May 2022 / Published: 5 May 2022
(This article belongs to the Section Biodiversity and Functionality of Aquatic Ecosystems)
Browse Figures
Review Reports Versions Notes

Abstract

:
The fate of pharmaceuticals in the human body, from their absorption to excretion is well studied. However, medication often leaves the patient’s body in an unchanged or metabolised, yet still active, form. Diazepam and its metabolites, ranging up to 100 µg/L, have been detected in surface waters worldwide; therefore, the question of its influence on model aquatic plants, such as duckweed (Lemna minor), needs to be addressed. Lemna was cultivated in a Steinberg medium containing diazepam in three concentrations—0.2, 20, and 2000 µg/L. The activity of superoxide dismutase (SOD) and catalase (CAT), leaf count, mass, and the fluorescence quantum yield of photosynthesis were assessed. The medium was also analysed by LC-MS/MS to determine the concentration of diazepam metabolites. Our results show no negative impact of diazepam on Lemna minor, even in concentrations significantly higher than those that are ecotoxicologically relevant. On the contrary, the influence of diazepam on Lemna suggests growth stimulation and a similarity to the effect diazepam has on the human body. The comparison to the human body may be accurate because γ-Aminobutyric acid-like (GABA-like) receptors responsible for the effect in humans have also been recently described in plants. Therefore, our results can open an interesting scientific area, indicating that GABA receptors and interference with benzodiazepines are evolutionarily much older than previously anticipated. This could help to answer more questions related to the reaction of aquatic organisms to micropollutants such as psychopharmaceuticals.

1. Introduction

The fate of different pharmaceuticals in the human body has been well studied [1]. However, only a small portion of the attention focuses on the fact that medication often leaves the patient’s body in unchanged or metabolised, yet still active, form, and if not properly removed at wastewater treatment plants, it enters the aquatic environment [2].
Worldwide, future water availability causes concern. According to the 2019 OECD Survey on the Implementation of the OECD Council Recommendation on Water, 22 out of 26 countries responded that their national water management plan consider uncertainties about future water availability and demand [3].
In 2000, the EU Water Framework Directive laid the foundations for Directive 2013/39/EU regarding priority substances in the field of water policy. According to the Directive, acute toxicity effects of chemical and pharmaceutical pollution of surface water and the chronic exposure and accumulation of chemicals in the environment pose a serious threat, not only to human health, but also to biodiversity and cause the loss of natural habitats [4].
The EU Member States reported that an average 48.6% of their population was using prescribed medicines in 2014, ranging from 22.8% in Romania to 60.2% in Belgium. This average clearly depends on age, ranging from 2.4% in the age group of 15 to 24 years in Romania to 96.3% in the age group of 75 years and over in the Czech Republic [5,6].
When it comes to non-prescription medicines, the average is 34.6% across the EU Member States, ranging from 15.2% in Romania to 84.4% in Iceland. The age dependency has, however, shifted when compared to prescription medicines, ranging from 9.8% in the age group from 15 to 24 years in Romania to 87.0% in the age group of 65 to 74 years in Iceland [6].
A range of different pharmaceuticals including diazepam has been reported in surface water [7], WWTP effluents [8,9], underground water [10,11], drinking water [12], fish tissue, and in the tissue of other aquatic organisms [13,14,15]. The most commonly detected pharmaceuticals are antibiotics (erythromycin, ofloxacin), antiinflamatory drugs/analgesics (diclofenac, ibuprofen), lipid regulators (benzafibrate, gemfibrozil), beta-blockers (metoprolol, propranolol), cancer therapeutics (cyclophosphamide, ifosphamide), diuretics (furosemide), antiepileptics (carbamazepine), antidepressants (mianserin), steroids and hormones (17β-estradiol, estrone), and tranquilisers (diazepam) [2].
Most of these groups of pharmaceuticals have been thoroughly studied in relation to the aquatic environment; however, the evidence on benzodiazepines, namely diazepam and its metabolites, is limited.
In humans, diazepam (DIA) binds to a specific benzodiazepine binding site on GABA receptors to mediate its sedative and anxiolytic effect by opening Cl− channels and hyperpolarizing the membrane of neuronal cells [16]. It is metabolised by the P450 cytochrome. Diazepam is demethylated into nordazepam (NOR, desmethyldiazepam) and further hydroxylated into an even more active form—oxazepam (OXA) (Figure 1) [17]. The hydroxylation of the diazepam molecule itself results in a second intermediate metabolite—temazepam (TEM), which is hydroxylated into oxazepam. Oxazepam is then glucuronised and leaves the human body in urine [18,19]. Diazepam and its metabolites ranging up to 100 µg/L have been detected in both fresh and saline surface waters worldwide [12,14,20,21,22] and have been marked as highly persistent in the sediment [23]. Diazepam has been shown to reduce motile response to light in Daphnia magna [24], decrease heart rate and delay hatching time of Danio rerio embryos, increase locomotion activity and reduce thigmotaxis of Danio rerio larvae [25], and reduce serotonin levels in Elliptio complanate [26]. The influence of the diazepam metabolite oxazepam on different aquatic organisms has also been studied, showing adverse effects connected to activity, sociality, boldness, and feeding rates [13,27,28].
Lemna minor, L., commonly called duckweed, is a globally cosmopolitan species of monocotyledons, which is reflected by the OECD by defining Test No. 221: Lemna sp. Growth Inhibition Test in the OECD Guidelines for the Testing of Chemicals [29]. It has been used as a model plant for many ecotoxicological tests, including tests of the toxicity of pharmaceuticals such as estrogens, ibuprofen, caffeine, and others [30]. Because Lemna is an aquatic organism, it is possible to precisely analyse the concentration of tested pharmaceuticals in the medium, including their eventual metabolites.
Moreover, Lemna is often a source of protein for ducks, fish, and other animals, which would allow the pharmaceuticals to enter the food chain in cases where they have been absorbed by the plant. Lemna is also used for phytoremediation and has been studied for its capacity to remove heavy metals [31,32]. This is why understanding the influence of pharmaceuticals is crucial.
The aim of the study is to investigate the subchronic effect of three different concentrations of diazepam (0.2, 20, and 2000 µg/L) on Lemna minor over a seven-day exposure and to assess the products of diazepam metabolism both qualitatively and quantitatively.

2. Materials and Methods

2.1. Chemicals

Non-labelled diazepam was purchased from Fagron (Rotterdam, Netherlands). Labelled diazepam-d5 and analytical standards of diazepam metabolites (oxazepam, temazepam and nordazepam) were purchased from Fisher Scientific (Walthman, MA, USA), diluted by methanol, and stored in the refrigerator at 8 °C. Dimethyl sulfoxide (≥99.9%), dichloromethane, ether, hexane, methanol, formic acid, and chemicals for the Steinberg medium (potassium nitrate, calcium nitrate tetrahydrate, potassium phosphate monobasic, potassium phosphate dibasic, magnesium sulphate heptahydrate, boric acid, zinc sulphate heptahydrate, sodium molybdate dihydrate, manganese(II) chloride tetrahydrate, iron(III) chloride hexahydrate, EDTA disodium-dihydrate, sodium hydroxide, hydrochloric acid) were purchased from Sigma–Aldrich (Walthman, MA, USA). Reactive oxygen species (ROS) kits (catalase and superoxide dismutase) were purchased from Invitrogen (Walthman, MA, USA).

2.2. Lemna Cultivation

Diazepam was dissolved in ≥99.9% dimethyl sulfoxide (DMSO) and further diluted in a Steinberg medium to achieve the selected concentrations of 0.2 µg/L, 20 µg/L, and 2000 µg/L, with a maximum of 1% DMSO. Three sets of black opaque plastic cups were cut to the height of 5 cm and filled with 40 mL of diazepam solution of the given concentration. Pure Steinberg medium-filled cups served as a control. One more set was used to control the natural photodegradability and sorption of diazepam without the presence of the plants.
Lemna minor was cultivated in Steinberg medium under laboratory conditions (12 h light-darkness cycle, white light 100 µmol/m2.s, provided by fluorescent lamps, at 22 ± 2 °C). Ten to twelve healthy leaves were selected, counted, documented, and transplanted into the prepared plastic cups containing the medium with or without the diazepam solution, beside the photodegradability and sorption control set.
The experiment was carried out over 7 days. At each timepoint of t = 0, 24, 72, 120, and 168 h, one cup from each set was removed, the leaves were counted and documented, and their photosynthetic activity was measured. The plants were then removed, dried carefully with a paper towel, weighed on analytical scales, and frozen in 2 mL Eppendorf tubes at −80 °C for no more than 3 months prior to the biochemical analysis. The remaining medium was collected into sterile 50 mL plastic tubes and stored at −80 °C for up to 8 months prior to the LC-MS/MS analysis.

2.3. Chlorophyll Fluorescence Measurement

Before the measurements, all cups containing Lemna were dark-adapted for 5 min and then scanned by the chlorophyll fluorescence imaging camera FluorCam MF 700 (Photon Systems Instruments, Drasov, Czech Republic). Slow Kautsky kinetics supplemented with saturation pulses was applied. During the procedure, the leaves were illuminated by short weak pulses of red-measuring light (0.5 µmol/m2.s) to record the basal chlorophyll fluorescence level (F0) followed by a strong pulse of white saturating light (0.8 ms, 2000 µmol/m2.s) to reach the maximum fluorescence level (FM). After a 30 s relaxation in the darkness, red actinic light (120 µmol/m2.s) was turned on for 3 min. After this light-adaptation period, a steady-state fluorescence level (Ft, Ft_Lss) was recorded and another saturation pulse was applied to reach FM’ in the light-adapted state. The chlorophyll fluorescence values recorded were then used for calculating a set of chlorophyll fluorescence characteristics, including the leaf surface area, which was assessed by pixel count performed directly by FluorCam software (PSI, Drasov, Czech Republic).

2.4. ROS Analysis

Frozen plant samples with 1 mL of ultrapure water were sonicated by impulse ultrasound on ice. The samples were centrifuged, and supernatant protein was quantified. Standard catalase (CAT) and superoxide dismutase (SOD) assays were performed and assessed as instructed by the manufacturer using a Tecan Spark® reader (Tecan Trading AG, Mannedorf, Switzerland).

2.5. Medium Extraction

One millilitre of the medium was extracted by 1 mL of dichloromethane-ether-hexane (30:50:20), spiked by 10 ng diazepam-d5, dried under nitrogen gas, and reconstituted by 1 mL of 50% methanol. All samples were immediately measured by liquid chromatography coupled with tandem mass spectrometer (LC-MS/MS).

2.6. LC-MS/MS Analysis

The concentrations of the target molecules in the samples were determined using LC-MS/MS. Benzodiazepines were detected and quantified by the Agilent 1260 Infinity high-performance liquid chromatography system (Agilent Technologies, Santa Clara, CA, USA) combined with the Agilent 6460 TripleQuad mass spectrometer (Agilent Technologies, CA, USA) equipped with the electrospray ionization interface (ESI). Separation was achieved using Poroshell 120 EC-C18 (2.1 × 50 mm; 2.7 µm) fitted with a security guard column of the same packing material (Agilent Technologies, Santa Clara, CA, USA). The mobile phase A and B consisted of 0.2% formic acid and methanol, respectively. The gradient used was: 0–10 min, linear gradient from 50% to 100% B; 10–13 min, held at 100% B followed by 4 min of re-equilibration before the next injection. The flow rate was 200 µL/min, and the injection volume was 2 µL. Mass data were acquired using MassHunter Workstation software (Agilent Technologies, Santa Clara, CA, USA) multiple reaction monitoring (MRM) in the positive mode. The detector settings were: capillary voltage 3000 V, nozzle voltage 300 V, gas temperature (N2) 200 °C, gas flow 6 mL/min, nebulizer 40 psi, sheath gas temperature (N2) 250 °C, sheath gas flow 10 mL/min. For the quantification and qualification of benzodiazepines, MRM transitions of protonated ions were used (see Table A1 of Appendix A).
To ensure quality, the instrument was calibrated daily with multi-level calibration curves. The detection limits were determined as a 3:1 signal-to-noise ratio (S/N) and were 0.02 µg/L for oxazepam and temazepam, and 0.01 µg/L for diazepam and nordazepam. Process efficiency (PE) and spiked recoveries (RE) determined according to the previously published method [33] differed for each metabolite and each concentration, and can be found in Table 1. The coefficients of variation ranged from 8.2 to 11.9% for oxazepam, from 5.8 to 10.3% for temazepam, from 4.6 to 9.5% for nordazepam, and from 3.3 to 7.5% for diazepam at three different target levels (0.20, 20, and 2000 µg/L).

2.7. Statistical Analysis

Data were evaluated using IBM SPSS Statistics software. Normal distribution in the samples was assessed using the Shapiro–Wilk normality test, and significance was assessed by Student’s t-test, comparing sample values against the control.

3. Results

From all three concentration levels, most of the effects were observed in plants treated by 2000 µg/L of diazepam, which is high above the ecotoxicologically relevant levels. However, some of the effects were detected even at lower concentrations, which correspond to diazepam levels already reported in surface waters worldwide.
The influence of diazepam in the form of oxidative stress was observed at the molecular level already on day 1. SOD synthesis was 16.36% higher in diazepam-treated samples on day 1 as compared to the control, and 9.39% lower than the control on day 2; however, differences were not statistically significant. On day 3, the changes were visible also physiologically—the leaf count increased by 8.18% in samples treated with the lowest concentration of 0.2 µg/L diazepam; however, a statistically significant change was observed only on day 7 in the highest concentration of 2000 µg/L. The maximum quantum yield of photosynthesis was significantly higher in the lowest diazepam-treated samples (0.2 µg/L) on day 7; however, the trend was observed in all concentrations. This suggests that the ecologically relevant concentrations of diazepam stimulate photosynthesis slightly and it proves that high concentrations of diazepam do not inhibit photosynthesis in Lemna minor. The first metabolites of diazepam (temazepam and nordazepam) were detected in the medium already on day 2, and their concentration increased over time. The specific mechanism of the metabolic degradation of the mother molecule is yet to be discovered, and the eventual bacterial degradation should also be considered.

3.1. Biochemistry

Superoxide dismutase levels were elevated on the first day after exposure and dropped below the control level on day 3, where they stayed for next 2 days before returning to the control level (Figure 2A).
Catalase synthesis was above the control levels starting on day 1, continued to grow between days 3 and 5, and returned to the control levels on day 7 (Figure 2B).

3.2. QYmax

The levels of QYmax ranged from 0.71 to 0.81 throughout the experiment (Figure 3A).
The Photosynthesis yield was affected on day 7 of the experiment and was statistically significant only in the samples treated with the lowest diazepam concentration (0.2 µg/L). No negative effect was observed, even at the highest diazepam concentration (2000 µg/L).

3.3. Fronds Growth

On day 7, the number of fronds significantly increased at c = 2000 µg/L (Figure 3B). For other days and concentrations, no significant effect was observed. In the control group, the average increase was 334.7%, while in samples treated with 2000 µg/L of diazepam, it was 372.2%.

3.4. Mass Growth

After seven days, the fronds biomass increased by 350% in the control and by 470.4% in the sample treated with 2000 µg/L diazepam. The mass per leaf ratio increased by 21.2% for the highest concentration of diazepam (Figure 3C,D). The plant leaf area was measured by PSI FluorCam FC 800-C and re-calculated per leaf. No statistical difference between the control and the treated samples was detected.

3.5. Metabolism

In the 0.2 µg/L diazepam-treated samples, the only metabolite detected was oxazepam, which appeared already on day 1 (Figure 4A). After 24 h, the amount of oxazepam started to decrease and was below LOD on day 5.
For the diazepam concentration of 20 µg/L, the first detected metabolite was temazepam on day 1 (Figure 4B), followed by nordazepam on day 3 (Figure 4C). The concentration of both metabolites kept growing throughout the test, as opposed to the control samples without the plants, where the metabolites were only detected on day 3 and kept decreasing. Oxazepam was not detected in the samples at all.
The concentration of 2000 µg/L exhibited a steady growth in both temazepam and nordazepam concentrations (Figure 4D,E). The detected levels of metabolites increased with a tendency which was steeper than in the control samples without the plants. No oxazepam was detected in the medium.

4. Discussion

4.1. Diazepam Concentration Levels

Currently, most studies are concerned with the benzodiazepine levels detected in surface waters worldwide. However, it is important to know where the effect threshold lies. This is why three diazepam levels were evaluated—the ecotoxicologically relevant concentration of 20 µg/L and 100× lower (0.2 µg/L) and 100× higher (2000 µg/L) concentrations. During the 7-day exposure, the synthesis of new leaves occurred and, therefore, semi-chronic effects could be observed in several life cycle phases of Lemna.

4.2. Biochemistry

The superoxide dismutase levels rose immediately on the first day after exposure and dropped below the control levels on day 3. Superoxide dismutase levels followed this trend for two more days before rising back to the control level on day 7. We hypothesize that diazepam interferes with oxidative stress and initially induces a superoxide dismutase synthesis and reduces the stress answer.
Catalase levels rose over the first five days, leaving more catalase available for oxidative stress reactions. The results suggest that diazepam reduces the stress answer in Lemna for the first five days before it decreases back to the control levels.

4.3. Photosynthesis

A statistically significant difference (p ≤ 0.05) in photosynthesis was observed only in the plants treated with the lowest concentrations of diazepam (0.2 µg/L) (Figure 3A). Because the standardized mean difference was not high, the effect seems to be minimal. Therefore, diazepam possibly does not interact with the response of plant photosystem II.

4.4. Physiological Changes

After seven days, samples treated with the highest concentration of diazepam exhibited a significantly higher fronds count, resulting in higher total biomass and area. When recalculated to mass per leaf ratio, the samples treated with the highest diazepam concentration also increased in mass, supporting the hypothesis that diazepam not only stimulates growth and frond division, but also increases the mass of each leaf. According to our observations, the area of individual fronds did not grow, meaning the fronds are thicker, but do not have a larger surface area as compared to the control.
Because the results show that diazepam in plants does not have a toxic effect at higher concentrations, and even exhibits stimulation, the underlying molecular mechanisms of action of diazepam and its metabolic pathways ought to be investigated. Previous studies reported GABA-like receptors in plants [34,35,36], and therefore the idea of a benzodiazepine-specific binding site in plant cells could be one of the answers.
In the past, several studies reported the effect of oxazepam on aquatic organisms, suggesting an effect on activity, sociality, boldness, and feeding rate (see Table 2.). Bourioug et al. reported no effect of oxazepam on Lemna plants in terms of frond number, fresh biomass, or frond area; however, the studied concentrations ranged from 0.5 to 50 µg/L and were administered in a mix containing other psychoactive compounds [37].

4.5. Metabolism

Quite recent studies suggest that plant metabolic pathways decompose diazepam in the same manner that the human body does. [38,39]. All three metabolites were detected in the medium (Figure 4). While the concentration of oxazepam in the medium without plants has increased steadily, this was not the case for the samples with Lemna biomass. The outcome suggests that oxazepam in the medium without Lemna is a result of photodegradation. As expected, the formation of oxazepam was only observed in the lowest concentration of diazepam, because the second phase of metabolism only follows the first phase. For any of the higher concentrations, no oxazepam levels were detected during the seven-day test, though the intermediate metabolites temazepam and nordazepam were detected, increasing every day. Arguably, when the metabolic pathways are busy with the first stage of metabolism, it takes longer than 7 days to degrade temazepam and nordazepam into oxazepam.
The precise metabolic pathways of diazepam in plants have not yet been described. In humans, the main effect is caused by the benzodiazepine-specific binding site in GABA receptors. Recently, GABA-like receptors have been found in plants; however, their precise structure remains unknown and so does the eventual presence of a benzodiazepine-binding site. Confirmation of an in-plant benzodiazepine-specific binding site in GABA-like receptors would be extremely scientifically interesting and could reveal the fact that, on a molecular level, psychopharmaceuticals such as diazepam can act similarly in animals as in plants.

5. Conclusions

Our research brings two important conclusions. Firstly, the influence of diazepam on Lemna minor suggests similarity to the human body—a reduction in oxidative stress markers, growth propagation, and identical metabolites. A comparison to the human body may be accurate because a similar form of GABA receptors responsible for the effect in humans has recently also been described in plants [40,41,42].
Secondly, fundamental findings relevant for freshwater phytoremediation technologies are presented, proving that even in high concentrations, diazepam does not exhibit acute nor subchronic adverse effects on Lemna minor’s growth, propagation, nor photosynthesis, but on the contrary, can slightly increase the biomass of Lemna.

Author Contributions

Conceptualization, B.M. and A.L.; methodology, A.L., E.M. and T.M.; validation, all authors; formal analysis, A.L., B.M., E.M. and T.M.; investigation, K.O., Š.Z., A.L., T.M. and P.P.; resources, B.M. and R.O.; data curation, A.L. and T.M.; writing—original draft preparation, A.L.; writing—review and editing, A.L., B.M. and T.M.; visualization, A.L.; supervision, B.M. and R.O.; project administration, B.M.; funding acquisition, B.M. and R.O. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Technology Agency of the Czech Republic, grant number SS01020006.

Data Availability Statement

Not applicable.

Acknowledgments

The authors wish to thank to the Faculty of Pharmacy, Masaryk University, and the Institute of Botany, Czech Academy of Science, for support.

Conflicts of Interest

The authors declare no conflict of interest.

Appendix A

Table
Table A1. LC-MS/MS parameters specific to each benzodiazepine.
Table A1. LC-MS/MS parameters specific to each benzodiazepine.
Parent ion(m/z)Quantifier/QualifierFragmentorCollision Energy (V)
diazepam285154/19315030/30
diazepam-D5290154/19815030/30
nordiazepam271140/16515030/30
oxazepam287241/10410030/40
temazepam301255/17710025/45

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Water 14 01484 g001 550
Figure 1. Diazepam metabolism. In the first phase, diazepam is metabolised into nordazepam by N-demethylation and into temazepam by hydroxylation. Further on, these are metabolised into oxazepam by hydroxylation or N-demethylation, respectively. Figure modified from [17].
Figure 1. Diazepam metabolism. In the first phase, diazepam is metabolised into nordazepam by N-demethylation and into temazepam by hydroxylation. Further on, these are metabolised into oxazepam by hydroxylation or N-demethylation, respectively. Figure modified from [17].
Water 14 01484 g001
Water 14 01484 g002 550
Figure 2. Biochemistry parameters of Lemna minor. Superoxide dismutase synthesis (A) increased immediately after 24 h, and it dropped below the control levels on days 3 and 5. The catalase level (B) remained elevated for the first five days after exposure before decreasing back to the control levels. The positive control was treated by 500 mg/L of potassium dichromate(VII) for 12 h. Data represent mean over 3 repetitions; standard deviations are indicated by whiskers.
Figure 2. Biochemistry parameters of Lemna minor. Superoxide dismutase synthesis (A) increased immediately after 24 h, and it dropped below the control levels on days 3 and 5. The catalase level (B) remained elevated for the first five days after exposure before decreasing back to the control levels. The positive control was treated by 500 mg/L of potassium dichromate(VII) for 12 h. Data represent mean over 3 repetitions; standard deviations are indicated by whiskers.
Water 14 01484 g002
Water 14 01484 g003 550
Figure 3. A stimulating effect on photosynthesis (A) was observed on the last day at the lowest concentration level of 0.2 µg/L of diazepam, while no negative effects were observed. A significant increase in fronds growth (B) was observed on the last day in plants treated with the highest diazepam concentration (2000 µg/L). The biomass of treated plants (C) increased by 470.4% by day 7, while the control group’s biomass only increased by 350%. Mass per leaf (D) was higher in the plants treated with 2000 µg/L of diazepam on the last day of the experiment, as compared to the control; lower concentrations were not statistically different from the control. Data represent the mean over 3 repetitions; standard deviations are indicated by whiskers. Statistical significance is marked according to the t-test p-values: * for p < 0.05, ** for p < 0.01, and *** for p < 0.001.
Figure 3. A stimulating effect on photosynthesis (A) was observed on the last day at the lowest concentration level of 0.2 µg/L of diazepam, while no negative effects were observed. A significant increase in fronds growth (B) was observed on the last day in plants treated with the highest diazepam concentration (2000 µg/L). The biomass of treated plants (C) increased by 470.4% by day 7, while the control group’s biomass only increased by 350%. Mass per leaf (D) was higher in the plants treated with 2000 µg/L of diazepam on the last day of the experiment, as compared to the control; lower concentrations were not statistically different from the control. Data represent the mean over 3 repetitions; standard deviations are indicated by whiskers. Statistical significance is marked according to the t-test p-values: * for p < 0.05, ** for p < 0.01, and *** for p < 0.001.
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Figure 4. Quantification of diazepam metabolites in the medium after 7 days of cultivation of Lemna and in the medium without Lemna. The highest concentration of oxazepam (OXA) in the medium of the plants treated with 0.2 µg/L of diazepam (A) was detected on day 1 and decreased below LOD by day 5. The concentration of oxazepam in the control cups without the plants kept growing until the last day of the experiment. No other metabolites were detected at this diazepam concentration level. For the medium of plants treated with 20 µg/L of diazepam, temazepam (TEM) (B) was detected on day 3 and was slowly increasing, as opposed to the control group without any plants. Nordazepam (C) followed the same trend. In the plants treated with the highest concentration of diazepam (2000 µg/L), the first metabolites were detected already on day 1, and their concentrations kept increasing rapidly (D,E).
Figure 4. Quantification of diazepam metabolites in the medium after 7 days of cultivation of Lemna and in the medium without Lemna. The highest concentration of oxazepam (OXA) in the medium of the plants treated with 0.2 µg/L of diazepam (A) was detected on day 1 and decreased below LOD by day 5. The concentration of oxazepam in the control cups without the plants kept growing until the last day of the experiment. No other metabolites were detected at this diazepam concentration level. For the medium of plants treated with 20 µg/L of diazepam, temazepam (TEM) (B) was detected on day 3 and was slowly increasing, as opposed to the control group without any plants. Nordazepam (C) followed the same trend. In the plants treated with the highest concentration of diazepam (2000 µg/L), the first metabolites were detected already on day 1, and their concentrations kept increasing rapidly (D,E).
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Table
Table 1. Sensitivity of detection, process efficiency and spiked recoveries of diazepam and its metabolites temazepam, nordazepam, and oxazepam in three different concentrations.
Table 1. Sensitivity of detection, process efficiency and spiked recoveries of diazepam and its metabolites temazepam, nordazepam, and oxazepam in three different concentrations.
OxazepamTemazepamNordazepamDiazepam
Concentration [µg/L]0.22020000.22020000.22020000.2202000
Process efficiency *102.99 ± 5.9677.89 ± 10.00103.59 ± 19.06111.15 ± 6.1486.00 ± 5.75104.56 ± 15.65104.74 ± 2.8185.10 ± 7.67104.10 ± 15.80134.76 ± 9.3882.90 ± 3.61102.05 ± 14.51
Spiked recoveries *113.58 ± 4.71102.76 ± 9.99124.23 ± 18.37115.76 ± 5.86109.05 ± 5.62122.98 ± 14.93113.50 ± 5.50110.00 ± 7.57121.29 ± 15.08110.14 ± 8.32105.15 ± 3.30119.57 ± 13.91
* data represent mean (%) ± standard deviation over 6 repetitions.
Table
Table 2. Effects of oxazepam on selected aquatic organisms.
Table 2. Effects of oxazepam on selected aquatic organisms.
OrganismCompoundDoseExposureEffectReference
Lemna minormixture of psychoactive drugs incl. oxazepam0.5 µg/L7daysNo effect on frond number, fresh biomass or frond area[37]
5 µg/L7days
50 µg/L7days
Corbicula flumineamixture of psychoactive drugs including oxazepam50 µg/L3 daysElevated EROD, GST, CAT, otherwise no effect
Perca fluviatilisoxazepam1.8 µg/L7 daysIncreased activity, reduced sociality[27]
910 µg/L7 daysIncreased activity, reduced sociality, increased boldness, higher feeding rate
Rutilus rutilusoxazepam280 µg/L-Increased boldness[28]
0.84 µg/L-Increased boldness and activity
Perca fluviatilisbenzodiazepine mix (OXA, BRO, TEM, CLO)9.1, 6.9, 5.7, 8.1 µg/L7 daysIncreased boldness and activity[13]
0.5, 0.5, 0.3, 0.4 µg/L7 daysIncreased boldness and activity
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Lamaczová, A.; Malina, T.; Maršálková, E.; Odehnalová, K.; Opatřilová, R.; Přibilová, P.; Zezulka, Š.; Maršálek, B. Anxiety in Duckweed–Metabolism and Effect of Diazepam on Lemna minor. Water 2022, 14, 1484. https://doi.org/10.3390/w14091484

AMA Style

Lamaczová A, Malina T, Maršálková E, Odehnalová K, Opatřilová R, Přibilová P, Zezulka Š, Maršálek B. Anxiety in Duckweed–Metabolism and Effect of Diazepam on Lemna minor. Water. 2022; 14(9):1484. https://doi.org/10.3390/w14091484

Chicago/Turabian Style

Lamaczová, Adéla, Tomáš Malina, Eliška Maršálková, Klára Odehnalová, Radka Opatřilová, Petra Přibilová, Štěpán Zezulka, and Blahoslav Maršálek. 2022. "Anxiety in Duckweed–Metabolism and Effect of Diazepam on Lemna minor" Water 14, no. 9: 1484. https://doi.org/10.3390/w14091484

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