Impacts of S-metolachlor and terbuthylazine in fatty acid and carbohydrate composition of the benthic clam Scrobicularia plana

https://doi.org/10.1016/j.ecoenv.2019.02.034Get rights and content

Highlights

  • S-metolachlor decreases highly unsaturated fatty acids in S. plana.

  • Terbuthylazine diminishes the fatty acid content and diversity of S. plana.

  • Terbuthylazine prejudices the most the nutritious quality of S. plana.

  • Muscles of S. plana exhibit superior fatty acid content that visceral masses.

  • S. plana releases glucose under chemical stress.

Abstract

S-metolachlor (SMOC) and terbuthylazine (TBA) are herbicides that commonly appear as active ingredients (a.i.) in the composition of plant protection products. In a previous work, experimental bioassays were performed using those chemicals to find suitable molecular biomarkers to assess its toxicity to the non-target species Scrobicularia plana. The results obtained showed that the pollutants produce mortality and biochemical changes at the species, namely in protein contents and enzymatic activity levels. Thus, for a better understanding of the total biochemical impacts of those pollutants in S. plana, the composition of fatty acids (FA) and carbohydrates (CH) of the survival organisms are investigated here. In addition, since this species is edible its biochemical profile is directly related to its nutritious quality, which is analysed in this study. Furthermore, the analyses were performed in two types of tissue - the muscle and visceral mass of each survival organism. The greatest changes in FA composition are observable in small size class, being the most sensitive size class both at the toxicological and biochemical level. FA contents are higher in small organisms, both at the field and under laboratory conditions, being the disparity between size classes higher in visceral masses than in muscles. Indeed, muscles adequately represent the FA profile since those molecules appear in higher content in this tissue compared to visceral masses, becoming the better indicator tissue of biochemical changes. Besides, using muscles, less amount of biomass is needed, so it turns out to be the most cost-effective tissue to be used as endpoint in future studies. FA profiles observed at SMOC and TBA exposure are different, organisms from TBA exposure presenting a lower nutritious quality, in terms of FA abundance and diversity, than the organisms exposed to SMOC. Still, SMOC produces reductions of HUFA, essential fatty acids that cannot be synthesized by the species. Moreover, HUFA (mostly EPA and DHA) occupied the greatest part of the FA composition of organisms exposed to the control treatments and to TBA; however, the decreases of HUFA caused by the SMOC exposure change the profiles and make SFA the most dominant group. These findings represent a risk of low occurrence of essential fatty acids in entire aquatic environments exposed to the chemicals studied. Regarding CH, glucose is the only monosaccharide found in S. plana which was expected since glycogen is the main polysaccharide in animal tissues. In general, the glucose content increases with a concentration of pollutants, whereas the glycogen concentration decreases, suggesting that the glucose is being released as a response to chemical stress. Thus, this work presents tools to assess biochemical impacts of S-metolachlor and terbuthylazine in aquatic systems and to goes deeper in the knowledge of these pollutants’ toxicity to non-target species to predict its propagation through aquatic trophic webs.

Introduction

Estuaries are complex systems commonly surrounded by farmland, residential and industrial areas, being under constant menace of anthropogenic pressures. Intensive agriculture practices are typical in those environments, leading to an overuse of fertilizers and pesticides. Indiscriminate applications of products for plant protection or production optimization eventually outcome in residues in non-target organisms. Around the Mondego estuary, located in Figueira da Foz (Portugal), there are around 15000 ha of cultivated land, producing mainly rice and corn. This circumstance represents a risk of organic contamination for the water quality and the aquatic communities.

The benthic clam Scrobicularia plana (da Costa, 1778), commonly known as the peppery furrow shell, has a wide geographical distribution as well as a high abundance at the Mondego estuary. S. plana is used as a food resource and is very appreciate by human-beings, owning a great economic value. This deposit-feeder species is able to filtrate organic pollutants and plays a key role in estuarine structure functioning since it cleans the freshwater column and influences the available food and the energy flow in the entire community, (Fossi Tankoua et al., 2013, Gonçalves et al., 2016, Gonçalves et al., 2017).

According to the agricultural cooperatives of the Mondego valley, Primextra® Gold TZ is the most-used herbicide in the cultivated land that surrounds the estuary. The main active ingredients (a.i.) of this product are S-metolachlor (SMOC), making 30% (w.w.), and terbuthylazine (TBA), making 18% (w/w) of the total volume of the commercial formula (Filimonova et al., 2016a, Gonçalves et al., 2016, Neves et al., 2015). Gonçalves et al. (2016) performed an ecotoxicological study with Primextra® Gold TZ reporting its lethal concentrations to S. plana and Cerastoderma edule. The main a.i. of this herbicide also appear in the composition of several other products for plant protection used worldwide, thus, in terms of risk assessment, it is important to understand how these chemicals produce toxicity individually, as well as joined in commercial formulas. Thus, in a previous work of our research team, two size classes of S. plana were exposed to growing concentrations of SMOC and TBA individually for 96 h, proving the occurrence of mortality and the higher sensitivity of the species to SMOC (LC10 – big size = 30.065, small size = 16.285) than to TBA (LC10 – big size = 46.284, small size = 35.988) (Gutiérrez et al., 2019), moreover, the species’ lower sensitivity to both a.i. individually than to the commercial formula – big size LC10 = 6.338 mg/L and small size LC10 = 2.206 mg/L – (values from Gonçalves et al., 2016) was further discussed in the cited study. Furthermore, changes in protein contents and enzymes’ activities responses to oxidative stress as a result of SMOC and TBA exposure were also reported in Gutiérrez et al. (2019). Accordingly, to broaden the comprehension of the ecotoxicological and biochemical impacts of these pollutants in S. plana, the analyses of the fatty acids (FA) and carbohydrates (CH) composition of the surviving organisms of those bioassays are accomplished in the present study.

SMOC is a chloroacetamide, known to interfere with fatty acid (FA) synthesis (Robert et al., 2007) by the inhibition of the elongase that produce the elongation of very long-chain fatty acids (VLCFAs) synthesis, inhibiting the expression of FAE1 gene (Neves et al., 2015). Besides, it is vastly documented that SMOC causes damage to non-target species from different trophic levels, such as reduced growth of amoebas (Amacker et al., 2018), low cell density in algae (Thakkar et al., 2013), mortality and low fecundity in crustaceans (Maazouzi et al., 2016, Neves et al., 2015), harmed early development in oysters and zebrafish (Gamain et al., 2017, Quintaneiro et al., 2017). On the other hand, TBA acts as a photosynthesis inhibitor, but has also been reported toxic for aquatic animals such as to cause steatosis in carp eggs (Velisek et al., 2016), alterations in structural cells of marbled crayfish (Velisek et al., 2017), cellular swelling and epithelial lifting in sea bass (Manera et al., 2016), bioaccumulation and impairments in liver detoxification of rainbow trout (Tarja et al., 2003). TBA's mode of action in animal cells is yet undescribed (Velisek et al., 2017), still, some proposals point to changes in the lipid composition of cell's membranes (Bermúdez-Saldaña et al., 2005). Therefore, FA profiling seems to be a proper approach to understand the biochemical effects of these pollutants, moreover, variations in FA abundance or diversity are considered as useful information to comprehend how chemical stress affects non-target species (Arts et al., 2009).

One of the major concerns about pesticides is their impact on primary producers in aquatic systems as well as their propagation through aquatic trophic webs. Variations in the FA composition of marine plankton and diatoms exposed to pesticides has already been reported (Filimonova et al., 2016a). Thus, the ability of S. plana to assimilate and transfer FA is key in aquatic systems due to its important trophic position, since this species eats primary producers while serves as food resource to consumer species, such as crustaceans, fish, wading birds and even human beings (Verdelhos et al., 2014, Verdelhos et al., 2015). The group of highly unsaturated fatty acids (HUFA), including eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA), is especially important since these FA are essential metabolites that are mainly synthesized by plants but not by humans, and only a few animals are able to convert them through elongation or desaturation of other FA with a low ratio between production and consumption (Brett and Muller-Navarra, 1997). Consequently, animal species must obtain those named “essential fatty acids” or EFA by feeding (Ladhar et al., 2014). The consumption of omega-3 and omega-6 is highly recommended since those FA are associated with the prevention of cardiovascular, inflammatory, ocular and neurological diseases, as well as the improvement of the cognitive and nervous systems development, among other benefits (Filimonova et al., 2016b). Bivalves are considered a rich source of FA, being very integrated in the Mediterranean diet, characteristic of South Europe. Unfortunately, there are studies on S. plana, a well-known font of EFA, that have proved that its FA composition is affected when exposed to stressors and its nutritious quality is consequently compromised (Gonçalves et al., 2016, Mesquita et al., 2018, Verdelhos et al., 2015).

The main and immediate energy source to cope with stress are CH, thus, their metabolism plays an important role in the maintenance of cell homeostasis during processes of immune defence. Theoretically, the presence of organic pollutants may impose high energetic costs for immune regulation resulting in an enhance of carbohydrate metabolism including glucose degradation, glycogenolysis, and gluconeogenesis (Lochmiller and Deerenberg, 2000). Then, the analysis of the carbohydrate composition of non-target species such as S. plana may offer relevant information about toxicants’ biochemical impacts.

Therefore, to broaden the knowledge of the toxicity of SMOC and TBA towards non-target aquatic species, the present study investigates the variations detected in the composition of fatty acids and carbohydrates of S. plana after being exposed to each chemical individually. Moreover, the analyses were conducted in two types of tissue - the muscle and the visceral mass – to determine the better indicator tissue to be used as an endpoint in future ecotoxicological studies with these chemicals.

Section snippets

Study area

The present study took place at the Mondego estuary, located in the Atlantic coast of Portugal near Figueira da Foz city (40°08′N, 8°50′W). The estuary is a mesotidal system, polyhaline (salinity varies between 18 and 30 psu), influenced by a tempered-warm climate. It has an area of 8.6 km2, approximately, and it comprises 2 channels (North and south), separated by the island of Morraceira, that joins again near the mouth. S. plana was captured at the Southern channel, that is shallow (high

Fatty acid profiles

In general, over the range of concentrations of SMOC and TBA, saturated fatty acid (SFA) increased and unsaturated fatty acid (UFA) decrease on small size class organisms (Table 1, Table 2, Table 3, Table 4). Visceral masses of small organisms, after maintained in a period of depuration, exhibit increases of SFA and HUFA (≈1% and 6.9%, respectively) and decreases in MUFA and PUFA (5% and 2.5%, respectively), when compared with quantifications in organisms from the field. In muscles the same

Discussion

The present manuscript highlights the impacts of the pesticides SMOC and TBA in the biochemical profile of S. plana. According to previous experimental studies, Primextra® Gold TZ and its a.i. produce toxic effects and change the lipidic composition of aquatic species of primary producers, such as Daphnia longispina, Thalassiosira weissflogii, Arcatia tonsa and Artemia franciscana (Filimonova et al., 2016a). Thus, to investigate these biochemical impacts in species from upper trophic levels

Conclusions

The present work reported that FA contents are higher in small organisms, both in the field and under laboratory conditions. Moreover, muscles properly represent the FA profile of complete organisms, since those molecules present higher content in this tissue than in visceral masses and there are no differences in terms of diversity, becoming the muscle the better and most cost-effective indicator tissue. FA profiles observed at SMOC and TBA exposure are different, with the organisms exposed to

Acknowledgements

This study was supported by Fundação para a Ciência e a Tecnologia (FCT) through the strategic projects UID/MAR/04292/2013 granted to MARE, UID/AMB/50017/2013 granted to CESAM, UID/CTM/50011/2013 and POCI-01-0145-FEDER-007679 granted to CICECO-Aveiro Institute of Materials, and UID/QUI/00062/2013 granted to QOPNA, when applicable co-financed by FEDER under the PT2020 Partnership Agreement. A.F.C. Mesquita, A. M. M. Gonçalves and C. Nunes thank FCT for the financial support provided through the

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