Assessing multigenerational effects of prednisolone to the freshwater snail, Physa acuta (Gastropoda: Physidae)

Highlights

• Continuous prednisolone (PDS) exposure caused generational impairments at multiple endpoints.

• Toxicity effects increased with the increase in exposure duration.

• Anomalies occurred in shell structure and size of exposed P. acuta.

• LOEC values (p < 0.001) for PDS exposure ranged from 32 to 4 μg/L in F0-F2.

• P. acuta is confirmed as a promising bioindicator to monitor GC pollution.

Abstract

Prednisolone (PDS), a potent synthetic glucocorticoid is widely prescribed for its exceptional anti-inflammatory properties. Several studies have detected the environmental presence of PDS in water bodies which has led to an ecological concern for its toxicity to non-target aquatic biota. The present study investigated the effects of exposure to PDS on different life-cycle stages and generations of the freshwater snail, Physa acuta. This continuous exposure over a period of multiple generations resulted in generational impairments at measured endpoints. LOEC values (p < 0.001) for PDS exposure ranged from 32 to 4 μg/L in exposed F0-F2 generations. Global DNA methylation (% 5-methyl cytosine) of adult progeny was found to be affected at higher test concentrations in comparison to the parent snails. Partially formed to completely missed growth components of shell structure and shell thinning in abnormally underdeveloped PDS exposed snails of F1 and F2 generation, was also observed in this multigenerational exposure experiment. The multigenerational study confirmed P. acuta as a promising bioindicator since critical effects of the long term glucocorticoid exposure opens up the way for further investigations on transgenerational toxicity in environmental toxicology and risk assessment and to monitor glucocorticoid pollution in aqueous ecosystem.

Introduction

Gastropods belong to an intensively studied taxonomic class within the phylum Mollusc, with nearly 100,000 recognized species residing in marine and freshwater habitats worldwide [1], [2]. Of these, the freshwater snail from the family Physidae, Physa acuta (Draparnaud, 1805), is a small left-handed and globally-invasive species most likely originating from North America [3] and presently having established populations in almost all continents except Antarctica [4], [5]. P. acuta have been well documented in several scientific studies because of their outstanding characteristics as a laboratory test organisms. Massive abundance, easy identification, limited mobility and short lifecycle made these snails a favourable test species in biomonitoring research with an added advantage of investigating generational exposure effects. Previous field and laboratory studies have established that P. acuta are sensitive to anthropogenic pharmaceutical contaminants causing multiple levels of physical and biological impairment or in extreme cases mortality [6], [7], [8], [9], [10], [11]. Ecologically, aquatic snails have been well-known to serve as a key link between primary producers and upper level aquatic species in aqueous food webs. It has been reported that the functional hormones for regulation of biological processes, growth and sex differentiation in molluscs are likely the vertebrate-like sex steroids such as testosterone and progesterone [12], [13], [14], [15], [16]. The steroidogenesis process is found to be managed by enzymatic action (17β-hydroxy steroid dehydrogenase, 3β-hydroxy steroid dehydrogenase and 5α-reductase) [17] and this pathway is carried via sequencing of estrogen receptor orthologs in the studied molluscs [18], [19]. Studies by Schwarz et al. [20], [21] showed that a common mussel Mytilus spp. displayed rapid and high capacity for a vertebrate steroid testosterone and estradiol-17 B from water.

Glucocorticoids (GC) are a well-recognised class of steroid hormones used extensively for their uncompetitive anti-inflammatory properties in comparison to their natural counterparts to remedy severe inflammation and regulate glucose metabolism, immune response and behavioural processes [22], [23], [24]. Prednisolone (PDS) a derivative of cortisol, is a widely prescribed synthetic GC which has a more predominant glucocorticoid activity than natural GCs [24], [25], [26]. The extensive consumption of PDS is of ecological concern due to its detection in environmental samples at concentrations ranged from ng/L to μg/L [27], [28], [29], [30], [31]. Wastewaters collected in Netherlands showed the presence of GCs in extracts of hospital wastewater collected prior to sewage treatment at levels up to 1918 ng/L PDS, 545 ng/L prednisone, 472 ng/L cortisone, 301 ng/L cortisol, etc. [27]. In France, effluents of an industrial WWTP (pharmaceutical manufacturing plant discharge) and receiving river water downstream samples showed high concentrations of GCs at levels up to 23 μg/L [28]. Water samples from the River Thames in the UK contained GCs at concentrations ranging between 30 and 850 ng/L [23] and effluent samples from wastewater treatment plants (WWTP) in the Arizona region of the USA had GCs at 16–24 ng/L [27]. A study by Chang et al. [30], reported the presence of PDS in the STP influents at concentrations ranging from 1.5 to 7.5 ng/L. PDS has been stated to be toxic to a number of non-target aquatic organisms and has been reported to induce osteoporosis-like phenotype in scales [32] and affecting embryogenesis process of zebrafish (Danio rerio) [33], negatively hamper the biological traits such as fecundity and survival in crustaceans (Ceriodaphnia dubia and Daphnia magna) [34], [35] and result in embryonic developmental abnormalities in gastropods [36]. Effects of PDS on aquatic snails were evaluated in our previous research which studied in detail the two lifecycle stages (egg masses and juveniles) of P. acuta, adversely affecting exposed test species and hampering embryonic and post-hatching development at nominal concentrations ranging from 62.5–125 μg/L [36].

The data obtained from the past work assisted us to continue the present multigenerational toxicity bioassays using environmentally relevant PDS nominal test concentrations, whereby multigenerational toxicity effects were hypothesised to be observed at multiple endpoints. Long-term responses to a toxicant have been considered important in ecotoxicology to study precise changes in species evolution and pollution dynamics. In addition, epigenetic inheritance was also studied in the present study that quantified the global DNA methylation content in the PDS-exposed adult snails of F_0adult and F_1adult generation. The epigenetic generational transmission of the suppressed methylation content through two exposed generations was investigated. A handful of studies have established that steroidal toxicants (such as glucocorticoids) have the potential to cause alterations in genetic material during short to long-term exposure bioassays, thereby resulting in generational toxicity transfer through an epigenetic inheritance [37], [38], [39], [40], [41]. Information on the presence of epigenetic phenomena such as global DNA methylation (%5mC content) in gastropods has been well established [42], [43], [44], [45], [46], [47], [48] but only a single multigenerational study is available that postulated any possible epigenetic alterations in the exposed species [44]. Muller et al. [44] presented an epigenetic effect of the endocrine-disrupting chemical vinclozolin on P. acuta, showing the presence and age-dependence of DNA methylation in the test species. To the best of our knowledge, there are no scientific studies available on the assessment of prednisolone (PDS) effects in a multigenerational bioassay regime, focussing on different lifecycle stages and generations of snails. Therefore, considering the ecological niche of P. acuta and the potential need to investigate the alterations in the phenotypic traits and global DNA-methylation of PDS- exposed species, the present multigenerational experiment was designed.