Ecotoxicology and Environmental Safety 204 (2020) 111082
Available online 11 August 2020
Eobania vermiculata as a potential indicator of nitrate contamination in soil
Rita Cofone, Federica Carraturo, Teresa Capriello, Giovanni Libralato, Antonietta Siciliano,
Carmela Del Giudice, Nicola Maio, Marco Guida, Ida Ferrandino
*Department of Biology, University of Naples Federico II, Via Cinthia 21, 80126, Naples, Italy
A R T I C L E I N F O Keywords: Nitrate contamination Survival Development Eobania vermiculata Bioindicator Soil A B S T R A C T
The effects of nitrates were analysed on the land snail Eobania vermiculata, a good bioindicator to assess the effects of certain pollutants in soil. It is known that the molluscs are very sensitive to contamination substances and can be used as sentinel organism for environmental pollution assessment. The nitrates are present in fer-tilizers and in food additives and their excess can not only be harmful to the environment but also dangerous for the humans. Indeed, in the mammals the nitrates are converted into nitrites and can cause a series of compli-cations as the formation of methaemoglobin and cancers. In this study, adult organisms of E. vermiculata were exposed to soil containing 2000 mg/L of nitrates for 30 days to evaluate the stool microbiome and the histo-logical changes at the level of the foot. Eggs of these snails were similarly treated to observe their hatching, survival and development. Histological changes were observed at level of the foot of adult snails exposed to nitrate and in their stools was evident an increase of bacteria, especially those that have a high ability to exploit nitrates and nitrogen as nutrients. Instead, the treated eggs showed changes in hatching, hypopigmentation of newborn snails and a decrease of their survival in time. The overall information obtained from these endpoints can provide important information regarding the quality of the environment. In addition, they also showed that the invertebrate organism E. vermiculata despite being a simple organism is very useful and efficient for eco-toxicological studies.
1. Introduction
Soil pollution is of great concern not only in urban and industrial areas but also in agroecosystem mainly due to fertilizers. A lot of sub-stances, such as heavy metals, for example, are very dangerous pollut-ants and for this reason their toxicity is being studied on different organisms, land animals as Podarcis sicula (Favorito et al., 2010, 2017;
Ferrandino et al., 2015) but also aquatic organisms (Favorito et al., 2011; Ferrandino and Grimaldi, 2008; Padrilah et al., 2017). There are polluting substances, even if present in reduced concentrations, which are in no way necessary for living beings such as cadmium (Ferrandino et al., 2009; Monaco et al., 2016, 2017a) and aluminium (Capriello et al., 2019; Monaco et al., 2017b), others that are necessary at low concentrations but become dangerous when their concentrations in-crease such as copper (Carotenuto et al., 2020) or nitrates (Keshari et al., 2016). Nitrate (NO3−) is considered an important macro-element
pro-moting macrophyte growth becoming mainly part of the nucleic acids, amino acids and proteins. Nitrate is a fundamental compound for plant metabolism and for the maintenance of ecosystems; some of its salts are
mainly used as organic and chemical fertilizers, they are also found in plant foods (Iammarino et al., 2011), food additives (E251 and E252) and are commonly used to season meat and other perishable products (Chetty and Prasad, 2016; Lerfall and Østerlie, 2011; Motta et al., 2019;
Sindelar and Milkowski, 2012; Waga et al., 2017).
The main sources of nitrate are associated with chemical fertilizers, animal organic waste effluents, dairy shed effluent and pond sludge, pig slurry, sewage sludge waste, and nitrate producing bacteria (Padrilah et al., 2017). High nitrate levels in the environment (e.g., soil and water sources) may result in human health problems, such as congenital de-fects or miscarriages in humans (Keshari et al., 2016), but also inducing methemoglobinemia and carcinogenicity (Fraser et al., 1980; Iammar-ino et al., 2011). The health and environmental concern with nitrate leaching have prompted social and political pressure like the Nitrate Directive 91/676/EEC (EEC, 1991) and the Water Framework Directive 2000/60/EC (WFD, 2000). The threshold limit for nitrates in drinkable water was set at 50 mg/L in the European Union (EEC, 1991; World Health Organization, 2011), while the United States Environmental Protection Agency (EPA, 2007) and the Water and Air Quality Bureau of
* Corresponding author.
E-mail address: ida.ferrandino@unina.it (I. Ferrandino).
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Canada (Health Canada, 2013) imposed the limit at 45 mg/L. Specif-ically, the Nitrate Directive focused on the preservation of waters against pollution caused by nitrates from agricultural sources enhancing proper agricultural practices as well. Thanks to this directive, the nitrate concentration in natural waters between 2004 and 2007, remained stable or decreased up to 70% in the monitored sites compared to 2000–2003.
Some studies have shown that the accumulation of nitrate leads to histological changes of the kidney and liver in rats (Basireddy et al., 2006; Samer et al., 2016). When the accumulation of nitrates occurs in ruminants, the excess in blood is absorbed in the digestive tract causing irritation of the intestinal mucosa or can get to the epidermis ( Iam-marino et al., 2011). Nitrate can influence the production of steroids in
Danio rerio and Salmo trutta (Bjerregaard et al., 2018) reducing the Ox-ygen Consumption Rate (OCR) of D. rerio embryos when injected into the chorion (Conlin et al., 2018).
Relatively few studies about nitrate toxicity are present in in-vertebrates. Alonso and Camargo (2003) showed that nitrate did not cause any mortality, but reduced the speed of movement (at 44.9, 88.8 and 156.1 mgN-NO3-/L) and the number of live newborns in Potamo-pyrgus antipodarum, a freshwater snail.
Snails are used for ecotoxicological studies because they are good model organisms (El-Khayat et al., 2015; El-Khayat et al., 2018; Itziou and Dimitriadis, 2012; Regoli et al., 2006), thanks to their distribution, easy sampling, stress tolerance and accumulation capacity of various pollutants (Berger and Dallinger, 1993; da Silva et al., 2013; Hamed et al., 2007; Hamlet et al., 2012; Heiba et al., 2002; Pe˜na et al., 2017;
Sharaf et al., 2015). Eobania vermiculata has many advantages being widespread in the Mediterranean region and used as food (Itziou and Dimitriadis, 2010, 2012). It is not an endangered species and it has been used in scientific research to evaluate the effects of some metals (Ali et al., 2015; Itziou et al., 2010; Mobarak et al., 2017), even if few studies are reported for histological investigations (Hamed et al., 2007).
E. vermiculata belongs to terrestrial gastropods, with almost 35,000
described species in the world (Barker, 2002). In snails, organic pol-lutants are accumulated mainly in the digestive gland (Dindal and Wurzinger, 1971) and in the foot (Pe˜na et al., 2017).
The aim of this study was to investigate the terrestrial gastropod
E. vermiculata as a potential model to evaluate soil contaminated by
nitrates considering a multi-endpoint approach including egg hatching, survival testing, adult histology and analyses stool microbiome. For this study, we used soil with nominal concentration of 2000 mg/L of nitrates obtained from KNO3. The exposure of organisms to nitrate and its salts
are not yet fully clear, and the use of a model as E. vermiculata could represent a valid alternative model to deepen the knowledge already known getting more information.
2. Materials and methods 2.1. Sample spiking and analysis
Commercial soil samples were spiked to a nominal concentration of 2000 mg/L of nitrates similarly to Sidhu et al. (2011). Nitrates were obtained from KNO3 (Sigma Aldrich, purity > 99.9%). A mother solution
of 3.5 g/L of KNO3 was prepared in physiological solution and added to
the reference soil considering a 1:10 w/v ratio (soil: solution). Nitrate concentrations were measured spectrophotometrically (Hach-Lange DR6000) according to (EPA-NERL, 1974) after 1, 15 and 30 days. An-alyses were carried out in triplicate. The starting concentration (2000 mg/L) is based on nitrate accumulation in fodder crops up to 150–2000 mg/L (Sidhu et al., 2011) and already used in denitrification studies in soils and waters contaminated by different nitrate sources (Cyplik et al., 2011; El-Shinnawi and Abeol-Naga, 1981).
2.2. Eobania vermiculata breeding and toxic test
Adult snails of E. vermiculata were collected from local gardens in Naples, Campania. Snails with an average weight of 4.7 g were used for our study. They were kept at temperatures of 24 ± 2 ◦C in laboratory and
divided into two groups, 3 controls and 3 treated, inside plastic con-tainers containing about 4 cm of soil. Before the treatment, the snails were kept in the laboratory for 2 weeks for the acclimatization. Snails were fed daily with lettuce leaves and watered to maintain humid conditions. Adults were exposed to soil treated with KNO3 for 30 days.
The collected snails have released 120 eggs before of the treatment. These eggs in part were treated on the day itself of the laying and others after a week. All eggs collected were distributed into soil jars; 60 eggs, laid for a week, were divided into six groups of 10 respectively, three of these groups used as control (groups Ctrl7) and the other three were exposed to the treatment (groups Nit7). Other 60 eggs laying that day were similarly divided, three group with 10 eggs for each were used as control (groups Ctrl1) and other three groups as treated (groups Nit1) for a total of 30 eggs per experimental group. The eggs (groups Nit1 and Nit7) were exposed to KNO3 and the treatment was continued after their
hatched for all their life. Snails development was followed under a Leica Zoom 2000 stereomicroscope, at a magnification of 10× and 20×, for all period of treatment. Survival, hatching time and morphological alter-ations were recorded and compared to that of controls.
2.3. Adult snail histology and histochemistry
At 30 days all the adult snails were anesthetized and drowned in 5% ethanol, placed in Bouin’s solution for 48 h, dissected, dehydrated and the foots of the all specimens were embedded in paraffin. Sections of 5
μm were processed by Hematoxylin-Eosin staining for the histo-
morphological study and PAS-reaction for the detection of the muco-polysaccharides in the all adult snails foots. The images were analysed and acquired by the light microscope (Zeiss, Germany).
2.4. Stool microbiome
Stool samples from snails incubated in negative control soil and ni-trate spiked soil, at environmental temperature, under natural direct sunlight, were collected (i.e. every 7 days for 4 times). Stool microbiome analysis included total bacterial count (TBC) and colony identification (Sanger sequencing). During the study, stool samples from snails incu-bated in negative controls soil and nitrate rich soil where collected. For stool samples collection, snails were transferred the day before to sterile containers, rinsed with sterile distilled water and kept in aseptic condi-tions; stools produced were collected and snails transferred to the orig-inal containers. Stool samples, thus taken, were analysed every week for four weeks and were submitted for microbiological evaluations. Samples collected after week 2 (days 8–14) and week 3 (days 15–21) (i.e. two weeks were considered sufficient to allow stool microbiome to adapt after exposure) were sent to molecular analysis, to identify and characterize microorganisms, using Sanger sequencing. The analysis was conducted from October 2019 to November 2019. The analysis of total bacterial count (TBC) was conducted following UNI EN ISO 7218:2013 standard-ized method: 1 g aliquots from samples were serially diluted in sterile plastic tubes containing 9 mL sterile deionized water. From each tube, 100 μL aliquots were spread plated on Plate Count Agar (PCA), incubated
for 72 h at 30.0 ± 2.5 ◦C. Colonies were counted and the results were
expressed in CFU/g. Bacterial count analysis was performed in triplicate, and the resulting values corresponded to the mean of the triplicates, evaluated on three 10-fold dilutions (1:10, 1:100, and 1:1000). Standard deviation and standard error were calculated for each sample; standard error bars were indicated in the resulting figure. Morphologically
different colonies were isolated and sub-cultured on PCA medium and underwent DNA extraction and amplification. Isolated colonies under-went DNA extraction: microbial DNA was amplified following extraction through denaturation at 98 ◦C for 10 min and supernatant recovery after
(Ivanov and Bachvarov, 1987). PCR reactions were conducted employing universal primers V3_f (5′-CCAGACTCCTACGGGAGGCAG-3′) and V6_r
(5′-TCGATGCAACGCGAAGAA-3′), complementary to the 16 S rRNA
gene V3 and V6 conserved regions (Chakravorty et al., 2007). PCR re-actions were carried out employing VWR Chemicals reagents: PCR Key Buffer Tripton, 10 μM dNTPs, 50 μM V3_f-V6_r primers, Taq polimerase,
added to 47 μL sterile deionized water, and 1,5 μL DNA. Incubation
conditions were 95 ◦C for 2 min initial denaturation, 35 cycles of 95 ◦C for
30 s, 62 ◦C for 30 s, and 72 ◦C for 30 s, 72 ◦C for 5 min final extension.
Templates were analysed on 1.5% agarose gel, stained with GelRed (Nucleic acid gel stain – BIOTIUM), compared to a 100 bp DNA ladder. PCR samples were sent to an external service for purification and sequencing. Data obtained were compared to NCBI Sequence Database sequences (Carraturo et al., 2016).
2.5. Statistical analysis
Hatching and survival data were checked for normality (Shapiro- Wilk (S–W)test) and homogeneity of variance (F-test) prior to the application of parametric methods (t Student’s test) to compare the potential significant differences between treatments and negative con-trols. If S–W or F-test failed, non-parametric methods were used (Mann- Whitney rank sum test). Hatching and survival percentages (vs time) were analysed for the best fit with parametric linear and non-linear models. The minimum level of acceptable significance was set at 0.05. Data were processed by means of GraphPad-Prism 7 (GraphPad Soft-ware, La Jolla California USA, www.graphpad.com).
3. Results and discussion 3.1. Nitrate concentrations
Nitrate concentration was measured after 1, 15 and 30 days: as ex-pected, values decreased from 2000 ± 14 mg/L (day 1), to 580 ± 2.5 mg/L (day 15), to 175 ± 2.5 mg/L (day 30) in the samples spiked with KNO3.
3.2. Hatching of eggs and survival of snails
Hatching of eggs were reported in Fig. 1A and B for Nit1 and Nit7, respectively. Data were normally distributed (S–W, p < 0.05, df = 12) and presented equal variances (F-test, p < 0.05, df = 12). According to the Student t-test, data distributions for both Nit1 and Nit7 were not significantly different from the respective negative controls’ distribu-tions (Ctrl1 and Ctrl7). Anyway, after day 15, both nitrate treatments reduced in a significant way (p < 0.05) snail eggs hatching compared to negative controls. Data distributions in Fig. 1A and B were best fitted (p
< 0.001, adjusted R2 = 0.999) to sigmoidal logistic four parameter curves (y = d + (a-d)/(1+(x/c)^b), where x = time, y = hatching %, a = the minimum value that can be obtained, b = Hill’s slope of the curve; c =inflection point, and d = the maximum value that can be).
For Nit1 group, 8 days of treatment, the 23% of the eggs hatched respect to the controls (Ctrl1) that started hatching only two days later (10 days after laying) with a rate of 13% (Fig. 1A). For Nit7 group on the day 6 of analysis (12 from the laying) the hatching rate was 30% compared to controls (Ctrl7) which was 17% (Fig. 1B). According to these results, the treatment acted on hatching times by anticipating them. In particular, Nit1 group showed a time of hatching shorter compared to that occurred in the other group.
The hatching then continued regularly for all groups. However, on the day 15, the maximum number of hatched eggs was reached for the Nit1 group, whereas for the Ctrl1 group, the maximum number was reached only on the day 20 of treatment. Furthermore, the number of hatched eggs for the Nit1 group was slightly lower than the total number of hatched eggs in the controls (Ctrl1). For Nit7 group, the maximum hatching was recorded after 15 days of treatment (21 days after laying) while the controls (Ctrl7) reached the maximum amount of eggs hatched on the day 18 of treatment. However, also in this case the number of hatched eggs of the controls was greater than that of the treated ones.
Survival data (from day 23) were shown in Fig. 2A and B for Nit1 and Nit7, respectively. Data were normally distributed (S–W, p < 0.05), but equal variance tests failed. Thus, we opted for the non-parametric Mann- Whitney rank sum test to evidence that Ctrl1 versus Nit1 (p < 0.05, df = 12) and Ctrl7 versus Nit7 (p < 0.01, df = 20) were significantly different according to their relative median values. Data distributions were best fitted considering a simple linear model (y = ax + b, where x = time, y = survival, a = angular coefficient, b = intercept). A significant decrease in
Fig. 1. Best fit of hatching (%) versus exposure time; (A) Ctrl1 (d = 81.6, b = − 8.6, c = 13.3, and a = − 0.4) and Nit1 (d = 73.4, b = − 3.6, c = 10.6, and a = 0). (B) Hatching (%) of groups Ctrl7 (d = 72.8, b = − 3.2, c = 7.2, and a = 0) and Nit7 (d = 101.1, b = − 2.8, c = 11.8, and a = 0); error bars refer to standard errors.
survival occurred since day 26 for eggs of Nit1 group (Fig. 2A) and, to a lesser extent, for the eggs exposed to treatment after one week of laid for group Nit7 (Fig. 2B). On day 37, end of the experiment, all organisms of Nit1 group were dead (Fig. 2A), values significantly higher than those registered in the control group with only 8% of death snails. Instead, Nit7 group showed a significant decrease of survival since day 26 (Fig. 2B). For this group, survival reached 0% after 60 days of treatment. Nitrate had a slower effect on the organisms treated after a week of deposition (Nit7) probably because when the treatment began, they were in an advanced stage of development compared to the Nit1 group. To the best of our knowledge there are no other case studies of exposure of Eobania vermiculata or other snails to nitrates showing impaired survival. Only Frakes et al. (1982) studied the growth and survival of juvenile and larval stages of anemonefish after exposing them to low and high levels of N-nitrate observing that survival and growth, were reduced at the highest exposure concentrations.
3.3. Development of snails
The development of E. vermiculata snails was followed for the dura-tion of their life, starting from eggs exposure. At day 28, digital pictures with the stereomicroscope were taken to highlight the developed al-terations. The snails both of groups Nit1 and Nit7, which were survived to the treatment until that day, showed changes in the shell compared to
control specimens (Fig. 3). The alterations observed in the treated snails consisted mainly in a hypopigmentation (Fig. 3B) compared to the controls (Fig. 3A). Moreover, the shells of treated appeared to be very fragile to the touch. These alterations remained constant even in sub-sequent treatment period and no new anomaly was detected. It is not known similar alterations induced by nitrate in other snails. However
Frakes et al. (1982), studying the effects at low and high levels of N-nitrate on juvenile and larval anemonefish, in addition to the reduc-tion in survival, he disclosed a delayed larval metamorphosis occurred both in the fish exposed to low nitrate system and of those exposed to high nitrate system. Little information is available on the effects and mechanism of nitrate on snails.
3.4. Histological changes
Exposed adult snails to nitrates (30 days) were used to assess the possible histological changes. The sections of ventral (VE) foot of control snails processed by Hematoxylin-Eosin staining showed a compact and well-preserved tissue with a thin outer layer epithelial. Moreover, diffuse, numerous and different glands (Gl) surrounded by connective tissue and muscle fibres were present in the inner sub-epithelial layer (Fig. 4A). The foot of treated snails showed evident tissue alterations respect to controls. Their outer epithelium appeared darker in color, all the tissue was more acidophilic and the glands were significantly
Fig. 2. Best fit of survival (%) versus exposure time; (A) Ctrl1 (a = − 0.5, b = 91.5) and Nit1 (a = − 4.0, b = 155.0) (p < 0.001). (B) Survival (%) of groups Ctrl7 (a = − 0.4, b = 91.4) and Nit7 (a = − 1.8, b = 113.8) (p < 0.001); error bars refer to standard errors.
decreased (Fig. 4B). Also in the dorsal area of the foot evident tissue differences were revealed by PAS reaction. In controls, the epithelial and the sub-epithelial layers appeared well organized and the PAS positivity was confined in the specific glands (MG) normal in shape and distri-bution (Fig. 4C). In the treated snails, the epithelium was markedly positivity to PAS reaction and the sub-epithelial layer showed an in-crease of the vacuolar spaces (VS) and a dein-crease of glands and of their PAS positivity (Fig. 4D). Snails move through movements of the foot and therefore their epithelial cells come into contact with numerous harmful substances (Janice et al., 2010; Tyrakowski et al., 2012). Several studies show that environmental pollutants have pathological effects on snails (Regoli et al., 2006). On the other hand, in conditions of pollution, it is known that molluscs are sensitive to the pathogenic effects of toxic substances, which in turn can lead to harmful changes in their immu-nological and physiological processes. In this study, darker coloring of outer epithelial layer could be due to increase of PAS-positive secretion (Ali and Said, 2019; El-Khayat et al., 2015) relating to the emptying of the glands (Greistorfer et al., 2017) and also to major number of cells, evidenced by a greater presence of nuclei (Fig. 4D). Indeed, cellular proliferation in the outer epithelial layer was also seen in other species of snail in contact with contaminated soil (Pe˜na et al., 2017). The snail foot is in fact the part more exposed to the toxic aggression of contam-inated soil. For this reason, the cellular proliferation and the increase of secretion, in this layer, could be related to a mechanism of defence.
In agree with our results, other studies showed that after exposure to stress, snails reported similar histological changes, such as narrowing of the unicellular glands secreting mucous, deeper folds, increased empty
2015; Pe˜na et al., 2017). Stress responses in invertebrates can occur as result of acute or chronic exposure to contaminated environments and, as such, the general health of individuals within such environments, both in terms of histopathological lesions and in the presence of infected organisms, can in definitively reflect the general health of these sites (Stentiford and Feist, 2005).
Fig. 4. E. vermiculata foot sections. (A, B): Hematoxylin-Eosin staining; (C, D): PAS-reaction. (A), ventral region of foot in the control snail: the epithelium (VE) shows well-defined margins and inner the sub-epithelial layer has many glands (Gl). (B), ventral region of foot in the treated snail: where the epithelium appears darker color and jagged and the sub epithelial layer shows a sensible reduction of glands compared to the controls. (C), dorsal region of foot in the control snail: PAS (+) is present in the specific mucipar glands (MG) and the tissue shows a regular organization with a clear differentiation of tissue layers and cellular types. (D), dorsal region of foot in the treated snail: a strong positivity to PAS staining is evident also in the epithelium (DE), the glands appear decrease with an increase of vacuolar spaces (VS) diffuse in the inner layer. Scale bars: 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web
3.5. Microbiological analysis of snail’s stools
Results regarding microbiological analysis report similar trends for negative control (NC) and nitrate spiked (NIT) samples. Total bacterial count (TBC) evaluation for negative sample (NC) showed a constant value in the first three weeks and a 1.5–2 log increase after 28 days. NIT sample registered a 1 log decrement after 1 week and TBC values were higher compared to NC counts during the experiments. Analysis were performed in triplicate (Fig. 5).
3.6. Molecular identification of stool microorganisms
Sanger sequencing analysis was performed on microorganisms iso-lated from snail stool samples at days 8–14 and days 15–21. Germs isolated from Negative Control (NC) samples are similar within the time period considered: Acinetobacter sp., Aeromonas sp., Kluyvera sp., and
Pseudomonas sp. were identified. Stool samples collected from snails
incubated in soil specimens spiked with nitrate evidenced the presence of a slightly different microbiota: beyond species from the genera
Aci-netobacter, Aeromonas, and Pseudomonas sp., Klebsiella sp. and Leclercia
sp. were characterized (Table 1).
Stool samples collected from snails incubated in soil spiked with nitrates evidenced a higher microbial count with respects to stools from snails incubated in negative control samples. The higher bacterial count may depend on the fact that nitrates constitute a growth source for microorganisms. The presence of strains that hold high ability of exploiting nitrates and nitrogen as nutrients was evidenced in stools sampled from snails incubated in soils rich in nitrates. The amendment of inorganic nitrogen was demonstrated in several researches reducing the abundance and the composition of soil microorganisms (Wang et al., 2017, 2018; Ling et al., 2017; Nie et al., 2018), although authors linked the reduction to limiting pH values, rather than nitrogen sources. Wang et al. (2018) analysed N and P inputs in the soil and the effects on bacterial abundance reporting the qualitative and quantitative decrease of microorganisms in soil treated with nitrates from 9 × 109 to 4 × 109
copies/g of dry soil, although soil pathways are different from the snail
metabolic processes. However, the same study further reported the significative increase of the relative abundance of Gammaproteobacteria in nitrate treated soils (Wang et al., 2018), confirming the dominance of the Gammaproteobacteria Class in response to high nitrate values, as resulted in the present research: Acinetobacter spp., Aeromonas spp.,
Leclercia spp., Klebsiella spp., and Pseudomonas spp., were indeed
iso-lated from snails stool. It may be possible that snail microbiota reacted to the higher nitrates concentration available in the soil, in favor of denitrifying germs, able to reduce nitrates to inorganic nitrogen. The outcome would confirm the hypothesis: in fact, microorganisms able to enhance nitrate reduction pathways were characterized, and the sub-stantial reduction of the nitrate concentration in the soil (a 70% decrease after 14 days, and a >90% decrease in 28 days) was registered. The microbial species characterized from snail stool samples agree with several researches aimed to characterize the gut microbiota of snails. Species from Acinetobacter, Aeromonas (A. caviae, in particular),
Enterobacter, Klebsiella (K. pneumoniae, in particular) Pseudomonas
genera were isolated from gut samples of the snails Achatina fulica and
Helix aspersa (Dar et al., 2017; Cardoso et al., 2012; Watkins and Sim-kiss, 1990; Charrier, 1990). Acinetobacter and Pseudomonas genera are capable of denitrification, through release of N2 or of nitrate reduction
to nitrite. In fact, the majority of Acinetobacter sp. strains are able to activate replication in mineral medium containing ammonium or nitrate salts and a single carbon and energy source such as pyruvate, acetate, or lactate: Acinetobacter beijerinckii, for example, employs acetate as the sole carbon source and ammonia as nitrogen source, although it is not able to enhance a dissimilative denitrification metabolism (Nemec et al., 2009). Acinetobacter lwoffii is an aerobic and non-fermentative micro-organism, which is ubiquitous, and thus a potential opportunistic pathogen in immuno-compromised hospital patients: it uses nitrogen sources but is not able to reduce nitrates (Wanger et al., 2017).
Aero-monas rivipollensis is a facultatively anaerobic, Gram-negative
bacte-rium, catalase-and oxydase-positive, able to reduce nitrates to nitrites; it is related to Aeromonas media and Aeromonas hydrophila (Marti and Balc´azar, 2015). Klebsiella pneumoniae generally colonizes the intestine, reaching other tissues of the organism (Paczosa and Mecsas, 2016): it
Table 1
Results of identification of isolated microorganisms from snail stools incubated in negative control and nitrate spiked soils. Analysis were conducted at week 2 and week 3.
Microorganism Max Score Total Score Query Cover E value % Identity Accession n.
Week 2 (d8-d14) – Bacteria isolated from Negative Control sample
Acinetobacter beijerinckii strain SH36 1282 1282 96% 0.0 99.57% MN093328.1
Aeromonas media strain L34 977 977 99% 0.0 93.07% KU179358.1
Aeromonas rivipollensis strain KN-Mc-11N1 1247 1228 100% 0.0 99.85% CP027856.1
Kluyvera intermedia strain HR2 1260 10084 100% 0.0 100.00% CP045843.1
Enterobacter hormaechei strain 1◦ 1186 1186 100% 0.0 96.77% LC487537.1
Pseudomonas soli strain CAU 1560 1295 1295 97% 0.0 99.58% MN538257.1
Week 2 (d8-d14) – Bacteria isolated from nitrate spiked sample
Acinetobacter lwoffii strain AsT13 339 678 65% 6,00E-89 94.27% KX866675.1
Aeromonas media strain 76C 977 977 99% 0.0 93.07% KU179358.1
Aeromonas rivipollensis strain SQ-5 1266 1266 99% 0.0 100.00% MN216272.1
Leclercia adecarboxylata 1264 1264 100% 0.0 99.71% AB931126.1
Pseudomonas aeruginosa strain NB2 1269 1269 100% 0.0 100.00% MN719036.1
Pseudomonas putida strain US5 1230 1230 100% 0.0 99.70% KX260958.1
Pseudomonas soli strain CAU 1560 1262 1262 100% 0.0 100.00% MN538257.1
Week 3 (d15-d21) – Bacteria isolated from negative control sample
Acinetobacter beijerinckii strain SH36 1282 1282 96% 0.0 99.57% MN093328.1
Aeromonas media strain 76C 977 977 99% 0.0 93.07% KU179358.1
Aeromonas rivipollensis strain KN-Mc-11N1 1247 1228 100% 0.0 99.85% CP027856.1
Kluyvera intermedia strain HR2 1260 10084 100% 0.0 100.00% CP045843.1
Pseudomonas soli strain CAU 1560 1295 1295 97% 0.0 99.58% MN538257.1
Week 3 (d15-d21) – Bacteria isolated from nitrate spiked sample
Acinetobacter lwoffii strain AsT13 339 678 65% 6,00E-89 94.27% KX866675.1
Aeromonas media strain L34 977 977 99% 0.0 93.07% KU179358.1
Aeromonas rivipollensis strain SQ-5 1266 1266 99% 0.0 100.00% MN216272.1
Klebsiella pneumoniae strain RCB288 1240 1240 100% 0.0 99.41% KT260500.1
can reduce nitrate, that can be expoilted as the sole nitrogen source for growth (Satoh et al., 1983). Kluyvera intermedia and Leclercia
ade-carboxylata, from the Enterobacteriaceae Family, are both able to use
nitrate, through its conversion to nitrite, using nitrate reductase (Pavan et al., 2005; Spiegelhauer et al., 2019). Pseudomonas genus in general, strains of Pseudomonas aeruginosa in particular, are able to use nitrate as a terminal acceptor of electrons under anaerobic conditions, through a denitrification metabolism, mainly employing nitrate or nitrite and can additionally ferment pyruvate and arginine, in absence of oxygen; furthermore, pyruvate fermentation can be inactivated by nitrate respiration (Eschbach et al., 2004). From preliminary data available, it can be hypothesized that the presence of high concentration of nitrate in soil is translated in changes in microbial composition of snails’ stool, promoting bacteria whose metabolisms is enhanced by the exploitation of nitrogen-based compounds.
4. Conclusion
In the present study, we showed that an invertebrate, the terrestrial gastropod Eobania vermiculata, is an alternative and optimum model to evaluate the contaminated soils by nitrates. The evidence here collected indicates that the nitrate targets several aspects and functions. The snails have proved to be an excellent model both when exposed to treatment as adults and in the early stages of development. Nitrate has been shown to induce a decrease in the survival of exposed organisms in the early stages of development. As regards the teratogenic effects, nitrate induced both hatching alterations and defects in the shell structure of the exposed organisms. The adults, on the other hand, investigated through histological stainings, showed tissue alterations of the foot, the most exposed part to the contaminated soil. Furthermore, a change was also observed in the total bacterial counts and the microbiota compo-sition of nitrate spiked snails compared to control. The collected data therefore confirm the usefulness of Eobania vermiculata as a valid alter-native model to study the contamination of soil by nitrate.
Author contributions
Rita Cofone: Conceptualization, Methodology, Writing - Original Draft; Federica Carraturo: Methodology, Validation; Teresa Capriello: Formal analysis, Writing – Original Draft; Giovanni Libralato: Formal analysis, Writing - Review & Editing; Antonietta Siciliano: Investigation; Carmela Del Giudice: Investigation; Nicola Maio: Methodology, Vali-dation; Marco Guida: Data Curation, Writing - Review & Editing; Ida Ferrandino: Supervision, Conceptualization, Writing - Review & Editing.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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