A multi-biomarker approach to assess toxicity of diclofenac and 4-OH diclofenac in Mytilus trossulus mussels - First evidence of diclofenac metabolite impact on molluscs.

Although the presence of pharmaceuticals in the environment is an issue widely addressed in research over the past two decades, still little is known about their transformation products. However, there are indications that some of these chemicals may be equally or even more harmful than parent compounds. Diclofenac (DCF) is among the most commonly detected pharmaceuticals in the aquatic environment, but the potential effects of its metabolites on organisms are poorly understood. Therefore, the present study aimed to evaluate and compare the toxicity of DCF and its metabolite, 4-hydroxy diclofenac (4-OH DCF), in mussels using a multi-biomarker approach. Mytilus trossulus mussels were exposed to DCF and 4-OH DCF at 68.22 and 20.85 µg/L (measured concentrations at day 0), respectively, for 7 days. In our work, we showed that both tested compounds have no effect on most of the enzymatic biomarkers tested. However, it has been shown that their action can affect the protein content in gills and also be reflected through histological markers. ENVIRONMENTAL IMPLICATION: Studies in recent years clearly prove that pharmaceuticals can negatively affect aquatic organisms. In addition to parent compounds, metabolites of pharmaceuticals can also be a significant environmental problem. In the present work, the effects of diclofenac and its main metabolite, 4-hydroxy diclofenac, on marine mussels were evaluated. Both compounds showed negative effects on mussels, which was primarily observed through histological changes. The present study therefore confirms that not only diclofenac, but also its main metabolite can have negative effects on aquatic organisms.

Ionic liquids as potentially hazardous pollutants: Evidences of their presence in the environment and recent analytical developments.

Ionic liquids (ILs) are considered to be very promising group of chemicals and the number of their potential applications is growing rapidly. However, while these compounds were originally proposed as a green alternative to classical solvents, there are certain doubts as to whether this classification is correct. Although in recent years there have been first reports published proving the presence of some ILs in the environment and even in human blood, at this point the scale of this possible problem is not yet fully understood. However, there is no doubt that as the number of ILs applications increases, analytical capabilities for rapid detection of possible environmental contamination should be also considered. Therefore, in this review paper, recent evidences for the ILs environmental contamination as well as analytical achievements related to the extraction of ILs from various environmental matrices have been summarized and important gaps and future perspectives have been pointed out. Based on the presented data it might be concluded that there is the urgent need for further development towards risk assessment of these potential environmental contaminants.

Long-term stability of diclofenac and 4-hydroxydiclofenac in the seawater and sediment microenvironments: Evaluation of biotic and abiotic factors.

Studies in recent years have shown that significant amounts of diclofenac (DCF) and its metabolites are present in marine coastal waters. Their continuous flow into the environment may be associated with numerous negative effects on both fauna and flora. Although more and more is known about the effects of pharmaceuticals on marine ecosystems, there are still many issues that have not received enough attention, but are essential for risk assessment, such as long term stability. Furthermore, interaction of pharmaceuticals with sediments, which are inhabited by rich microbial, meiofaunal and macrobenthic communities need investigation. Therefore, we undertook an analysis of the stability of DCF and its metabolite, 4-hydroxy diclofenac, in seawater and sediment collected from the brackish environment of Puck Bay. Our 29-day experiment was designed to gain a better understanding of the fate of these compounds under experimental conditions same as near the seafloor. Diclofenac concentration decreased by 31.5% and 20.4% in the tanks with sediment and autoclaved sediment, respectively during 29-day long experiment. In contrast, the concentration of 4-OH diclofenac decreased by 76.5% and 90.2% in sediment and autoclaved sediment, respectively. The concentration decrease of both compounds in the sediment tanks resulted from their sorption in the sediment and biodegradation. Obtained results show that marine sediments favour DCF and 4-OH DCF removal from the water column.

Transformation products of pharmaceuticals in the environment: Their fate, (eco)toxicity and bioaccumulation potential.

Nowadays, a huge scientific attention is being paid to the chemicals of emerging concern, which may pose a significant risk to the human and whole ecosystems. Among them, residues of pharmaceuticals are a widely investigated group of chemicals. In recent years it has been repeatedly demonstrated that pharmaceuticals are present in the environment and that some of them can be toxic to organisms as well as accumulate in their tissues. However, even though the knowledge of the presence, fate and possible threats posed by the parent forms of pharmaceuticals is quite extensive, their transformation products (TPs) have been disregarded for long time. Since last few years, this aspect has gained more scientific attention and recently published papers proved their common presence in the environment. Also the interest in terms of their toxicity, bioconcentration and stability in the environment has increased. Therefore, the aim of our paper was to revise and assess the current state of knowledge on the fate and effects resulting from the presence of the pharmaceuticals' transformation drugs in the environment. This review discusses the metabolites of compounds belonging to six major pharmaceutical groups: SSRIs, anticancer drugs, antibiotics, antihistamines, NSAIDs and opioids, additionally discussing other individual compounds for which literature data exist. The data presented in this paper prove that some TPs may be as harmful as their native forms, however for many groups of drugs this data is still insufficient to assess the risk posed by their presence in the environment.

Presence of pharmaceuticals and their metabolites in wild-living aquatic organisms - Current state of knowledge.

In the last decades an increasing number of studies has been published concerning contamination of aquatic ecosystems with pharmaceuticals. Yet, the distribution of these chemical compounds in aquatic environments raises many questions and uncertainties. Data on the presence of selected pharmaceuticals in the same water bodies varies significantly between different studies. Therefore, since early 1990 s, wild organisms have been used in research on environmental contamination with pharmaceuticals. Indeed, pharmaceutical levels measured in biological matrices may better reflect their overall presence in the aquatic environments as such levels include not only direct exposure of a given organisms to a specific pollutant but also processes such as bioaccumulation and biomagnification. In the present paper, data concerning occurrence of pharmaceuticals in aquatic biota was reviewed. So far, pharmaceuticals have been studied mainly in fish and molluscs, with only a few papers available on crustaceans and macroalgae. The most commonly found pharmaceuticals both in freshwater and marine organisms are antibiotics, antidepressants and NSAIDS while there is no information about the presence of anticancer drugs in aquatic organisms. Furthermore, only single studies were conducted in Africa and Australia. Hence, systematization of up-to-date knowledge, the main aim of this review, is needed for further research targeting.

Exposure of Mytilus trossulus to diclofenac and 4'-hydroxydiclofenac: Uptake, bioconcentration and mass balance for the evaluation of their environmental fate.

Diclofenac (DIC) is one of the most widely consumed drugs in the world, and its presence in the environment as well as potential effects on organisms are the subject of numerous recent scientific works. However, it is becoming clear that the risk posed by pharmaceuticals in the environment needs to be viewed more broadly and their numerous derivatives should also be considered. In fact, already published results confirm that the transformation products of NSAIDs including DIC may cause a variety of potentially negative effects on marine organisms, sometimes showing increased biological activity. To date, however, little is known about bioconcentration of DIC and DIC metabolites and the role of sex in this process. Therefore, the present study for the first time evaluates sex-related differences in DIC bioconcentration and estimates bioconcentration potential of DIC metabolite, 4-OH DIC, in the Mytilus trossulus tissues. In the experiment lasting 7 days, mussels were exposed to DIC and 4-OH DIC at concentrations 68.22 and 20.85 µg/L, respectively. Our study confirms that DIC can be taken up by organisms not only in its native form, but also as a metabolite, and metabolised further. Furthermore, in the present work, mass balance was performed and the stability of both studied compounds under experimental conditions was analysed. Obtained results suggest that DIC is more stable than its derivative under the tested conditions, but further analyses of the environmental fate of these compounds are necessary.

Metabolism of non-steroidal anti-inflammatory drugs by non-target wild-living organisms.

The presence of the non-steroidal anti-inflammatory drugs (NSAIDs) in the environment is a fact, and aquatic and soil organisms are chronically exposed to trace levels of these emerging pollutants. This review presents the current state of knowledge on the metabolic pathways of NSAIDs in organisms at various levels of biological organisation. More than 150 publications dealing with target or non-target analysis of selected NSAIDs (mainly diclofenac, ibuprofen, and naproxen) were collected. The metabolites of phase I and phase II are presented. The similarity of NSAIDs metabolism to that in mammals was observed in bacteria, microalgae, fungi, higher plants, invertebrates, and vertebrates. The differences, such as newly detected metabolites, the extracellular metabolism observed in bacteria and fungi, or phase III metabolism in plants, are highlighted. Metabolites detected in plants (conjugates with sugars and amino acids) but not found in any other organisms are described. Selected, in-depth studies with isolated bacterial strains showed the possibility of transforming NSAIDs into assimilable carbon sources. It has been found that some of the metabolites show higher toxicity than their parent forms. The presence of metabolites of NSAIDs in the environment is the cumulative effect of their introduction with wastewaters, their formation in wastewater treatment plants, and their transformation by non-target wild-living organisms.

In vitro methods for predicting the bioconcentration of xenobiotics in aquatic organisms.

The accumulation of anthropogenic chemical substances in aquatic organisms is an immensely important issue from the point of view of environmental protection. In the context of the increasing number and variety of compounds that may potentially enter the environment, there is a need for efficient and reliable solutions to assess the risks. However, the classic approach of testing with fish or other animals is not sufficient. Due to very high costs, significant time and labour intensity, as well as ethical concerns, in vivo methods need to be replaced by new laboratory-based tools. So far, many models have been developed to estimate the bioconcentration potential of chemicals. However, most of them are not sufficiently reliable and their predictions are based on limited input data, often obtained with doubtful quality. The octanol-water partition coefficient is still often used as the main laboratory tool for estimating bioconcentration. However, according to current knowledge, this method can lead to very unreliable results, both for neutral species and, above all, for ionic compounds. It is therefore essential to start using new, more advanced and credible solutions on a large scale. Over the last years, many in vitro methods have been newly developed or improved, allowing for a much more adequate estimation of the bioconcentration potential. Therefore, the aim of this work was to review the most recent laboratory methods for assessing the bioconcentration potential and to evaluate their applicability in further research.

Effects of environmentally relevant concentrations of diclofenac in Mytilus trossulus.

The presence of pharmaceuticals in the marine environment is a growing problem of global importance. Although awareness of the significance of this issue is increasing, many questions related to the ecotoxicology of pharmaceuticals remain unclear. Diclofenac is one of the drugs most commonly detected in the marine environment and its potential toxicity has been previously highlighted, thus its impact on organisms deserves a special attention. Therefore, in this study, a thorough analysis of the effects of diclofenac on a condition and tissue level of a model representative of marine invertebrates - Mytilus trossulus - was performed. During the 25-day experiment, divided into exposure and depuration phases, bivalves were exposed to two environmentally relevant drug concentrations of 4 and 40 µg/L. The study showed that mussels absorb diclofenac in their tissues and the highest recorded concentration was 1.692 µg/g dw on day 8. Moreover, the content of diclofenac metabolites (4-OH and 5-OH diclofenac) was also examined, but they were not detected either in water or in tissues. Although exposure to low diclofenac concentrations did not significantly affect the condition index of organisms, changes in numerous histopathological parameters were noted. Performed histological examination provided additional valuable information on the influence of drugs on the functioning of invertebrates. Nevertheless, applicability of histopathological techniques in ecotoxicology of drugs requires additional evaluation in future studies.

Toxic effects of NSAIDs in non-target species: A review from the perspective of the aquatic environment.

The presence of pharmaceuticals in the aquatic environment, both in marine and freshwater reservoirs, is a major concern of global environmental protection. Among the drugs that are most commonly used, NSAIDs tend to dominate. Currently, being aware of the problem caused by drug contamination, it is extremely important to evaluate the scale and the full spectrum of its consequences, from short-term to long-term effects. The influence on non-target aquatic animals can take place at many levels, and the effects can be seen both in behaviour and physiology, but also in genetic alterations or reproduction disorders, affecting the development of entire populations. This review summarises all the advances made to estimate the impact of NSAIDs on aquatic animals. Multicellular animals from all trophic levels, inhabiting both inland waters, seas and oceans, have been considered. Particular attention has been paid to chronic studies, conducted at low, environmentally-relevant concentrations, to estimate the real effects of the present pollution. The number of such studies has indeed increased in recent years, allowing for a better insight into the possible consequences of pharmaceutical pollution. It should be stressed, however, that our knowledge is still limited to a few model species, while there are many groups of organisms completely unexplored regarding the effects of drugs. Therefore, the main aim of this paper was to summarise the current state of knowledge on the toxicity of NSAIDs in aquatic animals, also identifying important gaps and major issues requiring further analysis.

Mytilidae as model organisms in the marine ecotoxicology of pharmaceuticals - A review.

Growing production and consumption of pharmaceuticals is a global problem. Due to insufficient data on the concentration and distribution of pharmaceuticals in the marine environment, there are no appropriate legal regulations concerning their emission. In order to understand all aspects of the fate of pharmaceuticals in the marine environment and their effect on marine biota, it is necessary to find the most appropriate model organism for this purpose. This paper presents an overview of the ecotoxicological studies of pharmaceuticals, regarding the assessment of Mytilidae as suitable organisms for biomonitoring programs and toxicity tests. The use of mussels in the monitoring of pharmaceuticals allows the observation of changes in the concentration and distribution of these compounds. This in turn gives valuable information on the amount of pharmaceutical pollutants released into the environment in different areas. In this context, information necessary for the assessment of risks related to pharmaceuticals in the marine environment are provided based on what effective management procedures can be developed. However, the accumulation capacity of individual Mytilidae species, the bioavailability of pharmaceuticals and their biological effects should be further scrutinized.

Evaluation of bioconcentration and metabolism of diclofenac in mussels Mytilus trossulus - laboratory study.

Interest in the presence of pharmaceutically active compounds in the aquatic environment has been growing for over 20 years, yet very few studies deal with the metabolism of pharmaceuticals in marine organisms. In this study, the bioaccumulation under short-term conditions and metabolism of diclofenac were investigated. Mytilus trossulus was used as a representative of the Baltic benthic fauna. The mussels were exposed to diclofenac at a concentration of 133.33 µg/L for five days, following a five-day depuration phase. The highest concentration of diclofenac (7.79 µg/g dw) in tissues was determined on day 3. Subsequently, the concentration of diclofenac in tissues decreased rapidly to 0.86 µg/g dw on day 5. After five days of depuration, the concentration of diclofenac was 0.21 µg/g dw. Hydroxylated diclofenac metabolites were found both in tissues of mussels and water. This study shows that M. trossulus has the potential to accumulate diclofenac and metabolize it to 4-OH and 5-OH diclofenac.