Effects of heavy metals copper and mercury on the biochemical composition and activity pattern of selected enzymes of molluscs: a review

Heavy metals that enter the aquatic environment pose a serious threat to the freshwater biota due to their general toxicity, highly persistent nature, tendency to bioconcentrate and bioaccumulate in an organism, and food chain amplification (Coetzee et al. , 2002; Ursinyova & Hladikova, 2000; Weis & Weis, 1977). lthough copper is an essential trace element required by all life forms, it becomes toxic at high concentrations. Mercury, which has no known metabolic function, is known to be the most toxic of all heavy metals.

The effects of heavy metals on aquatic organisms may manifest at all levels of biological organization. Heavy metals cause severe metabolic, biochemical, physiological, cellular, molecular, and structural impairments in animals. Proteins, carbohydrates, and lipids are the “building blocks of life,” providing energy for maintaining the architecture of the tissues in the body. Tissues of all organisms contain enzymes. Phosphatases, which dephosphorylate substances, have a variety of roles in cellular signaling and regulation. cid phosphatase ( CP), which is associated with the lysosome (Cheng & Rodrick, 1975) and occurs ubiquitously among animal and plant species (Hollander, 1971), plays an important role in mineral metabolism and metal detoxification. lkaline phosphatase ( LP) is a plasma membrane-bound enzyme (Ciro et al. , 1975) involved in membrane transport, glycogen synthesis (Gupta & Rao, 1974), protein synthesis (Pilo et al. , 1972), the synthesis of some enzymes (Summer, 1965), and secretory activity (Ibrahim et al. , 1974). minotransferases such as aspartate amino transferase ( ST) and alanine amino transferase ( LT), formerly called glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT), that catalyze the process of biological transamination, serve as a major link between protein and carbohydrate metabolism (Lehninger, 1979).

Mollusca, the second-largest metazoan taxon, consist of placophora, Polyplacophora, Monoplacophora, Gastropoda, Cephalopoda, Bivalvia, and Scaphopoda. (Ponder & Lindberg, 2008). It exhibits a wide range of diversity, representing about 50,000 to 1,20,000 living species (C hapman, 2009 ) and 70,000

fossil species (Brusca & Brusca, 2003; Rosenberg, 2014). Molluscs are considered a potential biomonitor for heavy metal contamination in aquatic ecosystems because of their widespread geographical distribution, abundance, availability, sedentary nature, suitable dimensions, size, and weight, ease of handling, collection, identification, and culture, relatively longer life span, high tolerance to environmental changes and persistent toxic chemicals, disease resistance, and high accumulation rates for the metals.

This review provides an overview of the effects of heavy metals (copper and mercury) on biochemical constituents such as protein, carbohydrate, lipid, and the activity pattern of enzymes such as acid and alkaline phosphatases, aspartate, and alanine aminotransferases in molluscs.

EFFECTS OF COPPER AND MERCURY ONTHE MOLLUSCS

Effects of copper on biochemical composition of bivalve

Katticaram et al. (1995) noticed a decline in total carbohydrate level in the digestive glands of the bivalve Sunetta scripta after prolonged exposure to higher concentrations of copper (0.3 to 1 ppm) and significantly higher protein levels at 0.3 ppm and 0.5 ppm copper. Exposure of the European freshwater mussel Anodonta anatina to Cu via water (0.3 µmol/L ⁶³Cu) or food (⁶³Cu-loaded algae, equivalent to 0.06 µmol/L) for 24 days results in a decrease in carbohydrate and protein levels in all body compartments (Nugroho & Frank, 2012). Sathyanathan et al. (1988) evaluated the effect of sublethal amounts of copper and mercury on the biochemical constituents of the estuarine clam Villorita cyprinoides over a range of time and described the changes in glycogen contents and metabolic rate of the tissues of the clam. Satyaparameshwar et al. (2006) recorded a decrease in levels of glycogen and a shift in the carbohydrate metabolism from aerobic to anaerobic type in selected tissues of the freshwater mussel Lamellidens marginalis due to the sublethal toxicity of copper sulfate. Neuhoff (1983) conducted a study on the synergistic physiological effects of low copper and various oxygen concentrations on the marine bivalve

Macoma balthica and concluded that the glycogen content of the tissues and their dry weight are more affected by a combination of oxygen deficiency and a low copper concentration (2.5 cm³ O 2 dm^-3, 15 μg Cu dm³). Gills and hepatopancreas of the freshwater mussel, Lamellidens corrianus , showed a general trend of depleted levels of glycogen activity at all time periods (24, 72, 120, and 168 h) and in all the exposures to copper (100, 200, and 400 ppb) and mercury (75, 150, and 300 ppb) when compared with the controls (Rajalekshmi & Mohandas, 1993).

In an experiment, Fokina et al. (2013) detected major changes in the gill and digestive gland lipid composition of blue mussels, Mytilus edulis , after copper and cadmium exposure. significant reduction in the activities of glycogen, protein, and lipid was found in the gill, mantle, gonads, and hepatopancreas of mercury chloride-treated Lamelledis marginalis (Muniv et al ., 2020). Sonawane (2015) confirmed that the total lipid content of the whole body, foot, and mantle decreased and digestive glands increased significantly in the bivalve Lamellidens marginalis after acute and chronic exposure to copper sulfate.

Miller (1988) assessed the interactive effects of copper exposure at various salinities on the biochemical responses of the ribbed mussel Geukensia demissa and pointed out impairments in biochemical functions. Significant differences in biochemical constituents’ activity were found when the sian green mussel Perna viridis was exposed to copper, cadmium, lead, and zinc concentrations under a long-term chronic toxicity test (Rajkumar & Milton, 2011).

Effects of mercury on biochemical composition of bivalve

Devi (1996) investigated the effect of mercury on the biochemical composition of the marine fouling dreissinid bivalve Mytilopsis sallei and concluded that in timedependent experiments, carbohydrates were utilized and a decrease in the ratios of glycogen to protein and glycogen to lipid was observed. In concentrationdependent exposure, both carbohydrates and proteins were consumed, and there was also a decrease in the glycogen to lipid ratio, but the glycogen to protein ratio was almost constant at all exposure concentrations. Reddy et al. (1986) concluded that the changes in the levels of glycogen in the foot, mantle, and gills of a freshwater mussel, Parreysia rugosa, under sublethal concentrations of mercury chloride indicate a decrease in energy supply metabolism through oxidative pathways. Sonawane (2018) noted greater depletion of average glycogen content in the whole body and digestive gland as compared to the foot and mantle of the bivalve Lamellidens marginalis when exposed to HgCl2 under acute and chronic treatment. significant glycogen decrease was documented by Bhamare et al. (2001) in the whole body, followed by the digestive gland, foot, and mantle of Parreysia favidens exposed to 3.24 mg/l mercuric chloride for 72 hours as acute treatment and 1.89 mg/l mercuric chloride for 20 days as chronic treatment. Pardeshi and Zambare (2012) found the depletion of glycogen contents in the mantle, foot, gills, gonads, and digestive glands of the bivalve Parreysia cylindrica due to acute (0.6 ppm) and chronic (0.12 ppm) doses of mercury chloride.

Ravinder (1991) indicated a decrease in levels of total proteins and an increase in free amino acids and proteolytic activity in the foot, mantle, and gill of the mussel Parreysia rugosa under the influence of sublethal concentrations of mercuric chloride (0.5 ppm). Protein contents were decreased in the gills, testis, and digestive glands of bivalves Lamellidens corrianus after acute exposure to mercury (0.444 ppm Hg) as compared to those of control bivalves (Gulbhile & Zambare, 2013). Suryawanshi et al. (2014) noticed a significant decrease in protein content in the mantle, adductor muscle, gonad, siphon, gills, hepatopancreas, and mantle of oysters, Crassostrea cattuckensis , under 0.01 ppm and 0.04 ppm mercury exposure for 15 days.

Effects of copper on the biochemical composition of gastropod

In a study carried out by Brown et al. (2004), significant effects were measured in the total protein and lysosomal stability of the hemolymph of the common limpet snail Patella vulgata and the blue mussel Mytilus edulis at a copper concentration of 6.1 μg/L. The high protein content change was found in the digestive gland, followed by the foot and mantle, in the freshwater snail Thiara tuberculata exposed to 5.45 and 1.14 ppm of copper sulfate as acute and chronic treatments, respectively (Mule & Lomte, 1995). Kumar et al. (2011) determined that the snail L. acuminata fed with sub-lethal doses, i.e., 20% and 60% of the 24 h and 96 h LC 50 of different molluscicides inside snail attractant pellets, caused a significant decrease in proteins and free amino acids in the ovotestis of the snail. Rawi et al. (2011), when exposed the snail B. alexandrina to sublethal doses (LC 10 ) of CuSO 4 and plant extract, observed a reduction in the total protein and total lipid contents in the snail’s hemolymph. significant decrease in soluble proteins, along with an insignificant decrease in structural proteins and a significant increase in free amino acids and protease activity, was observed by Johnson (1990) in the hepatopancreas and foot of the snail Pila globosa exposed to a lethal concentration of copper.

Wang et al. (2009) learned that the crude lipid in the carcass of juvenile abalone, Haliotis discus, decreased with increasing dietary copper and was significantly lower in abalone fed dietary Cu ≥ 26.84 mg/kg.

Effects of mercury on the biochemical composition of gastropod ccording to Mahajan (2005), the heavy metal mercury induced an alteration in the protein content of the gastropod Bellamya bengalensis. In the study conducted by Lomte and Sontakke (1992) on snails Thiara lineata, during a 24-hour exposure to mercuric chloride (2 ppm), the foot, mantle, and digestive gland showed an increase in protein content and during 48-, 72-, and 96-hour exposures, a considerable decrease in protein was observed in the foot, while an increase in protein was noticed in the mantle and digestive gland. Patil et al. (2011) noted that a sublethal concentration of mercuric chloride (0.1 ppm) showed a significant decrease in the level of glycogen, an increase in the level of protein, and an insignificant decline in the level of lipids in the sian snail Indoplanorbis exustus.

Mule and Lomte (1993) evaluated the effect of mercury chloride on the total lipid content of Thiara tuberculata and depicted that in chronic treatment the lipid contents of the foot and the mantle were increased while those of the digestive gland were decreased, and in acute treatment the lipid contents of the digestive gland and foot were decreased while those of the mantle were increased.

Effects of copper and mercury on the activity of phosphatase and aminotransferases of bivalve

Suresh and Mohandas (1990a) stated a high level of acid phosphatase in the hemolymph of the bivalve Sunetta scripta exposed to 1 ppm copper and a lower value at higher concentrations. ccording to Jing et al. (2007), copper treatment affected the activities of an immune enzyme ( CP) in the mantle of Pinctada fucata . In research by nthony and Patel (2000), exposure of the tropical arcid blood clam Anadara granosa to sublethal concentrations of Cu at different salinities inhibited the activities of the acid phosphatase. Rajalakshmi and Mohandas (2005) demonstrated variation in CP activity in the gills and hepatopancreas of the mussel Lamellidens corrianus exposed to different concentrations of copper for 24, 120, and 168 h. reduction in the latency of lysosomal enzymes and changes in the composition of copper-binding proteins were detected in the digestive gland cells of Mytilus edulis exposed to increased sublethal copper concentrations (Harrison, 1983). While studying the effects of copper (0.05 μM and 0.5 μM) on the activity of an enzyme involved in immune defense ( CP) and a metal-sensitive enzyme ( LP) in the gills and digestive gland of the pear oyster, Pinctada fucata , at 12, 24, 48, and 72 h of exposure, Jing et al . (2006) observed the altered activities of phosphatase by copper. Research by Sharma et al. (2006) on the green mussel Perna viridis exposed to 25 pg/l of copper and mercury

(sampling on days 0, 7, and 14) depicted higher CP activity in the digestive gland cells of treated mussels. The results of the study carried out by Sharma and Thomas (2007) revealed that the sublethal concentration (25 μg/l) of copper and mercury at 24 h, 72 h, the 7^th day, and the 14^th day can induce remarkably higher acid phosphatase activity (hyper synthesis) in the hemolymph of green mussels ( Perna viridis ), but on continued exposure, the CP activity shows a considerable decline. Regoli and Principato (1995) carried out analyses on the digestive gland and gills of mussels ( Mytilus galloprovincialis ) exposed to copper under field and laboratory conditions and concluded that, compared to control mussels, mussels transplanted into the polluted environment and mussels exposed to copper under laboratory conditions showed significant variations in the alkaline phosphatase enzyme. The mercury showed the inhibitory effects on acid and alkaline phosphatase activities in various tissues (digestive gland, gills, foot, siphon, and mantle) analyzed in the clam Scrobicularia plana (Mazorra et al., 2002).

Ravinder (1991) recorded a rise in the levels of amino acid metabolizing enzymes ( ST and LT) in the foot, mantle, and gills of a freshwater mussel, Parreysia rugosa , under the influence of sublethal concentrations of mercuric chloride (0.5 ppm). Kulkarni and Kulkarni (1987) indicated elevated levels of transaminase in the gills and mantle of the clam Katelisia opima when exposed to mercury.

In a study by Ramalingam and Indra (2002), the change in phosphatase activity in the body mass muscle, foot muscle, digestive gland, and hemolymph of Achatina fulica under sublethal toxicity of copper sulfate was recorded. Li et al. (2009) examined the toxicity of copper (1.35-4.20 mg/L) on the activities of the metabolic enzymes of the marine gastropod Onchidium struma after a 1-week exposure and detected an increase, then a decrease, and, finally, a slight increase in acid and alkaline phosphatase activity in the hepatopancreas, while aspartate and alanine transferases were activated by lower concentrations of

Cu and inhibited at higher concentrations in the hepatopancreas and muscle. Suresh et al. (1993) experimented on the effects of copper (0.010, 0.015, and 0.020 ppm) on the activity of phosphatases and transaminases in the hemolymph of the snails Indoplanorbis exustus , Lymnaea acuminata, and F. rufescens at 2, 6, 12, 24, and 48 h post-exposure and concluded that copper can cause hyper synthesis of lysosomal enzymes and, to a certain extent, can inhibit the activity of both enzymes.

Masola et al. (2003) mentioned that the activities of ST rose by up to 4.7-fold in the homogenate and the mitochondrial and cytosolic fractions with increasing concentrations of copper; these activities, however, fell at copper concentrations of approximately 1 mg/L, which coincided with the massive deaths of the snails Helisoma duryi and Lymnaea natalensis . Ragab (2003) presented a marked increase or inhibition in the activity of either ST or LT in the digestive gland of the three forms of snails, Egyptian Biomphalaria glabrata, by CuSO 4 . Rawi et al. (2011) showed that copper sulfate affected the activity of transaminase enzymes in the hemolymph of the snail B. alexandrina .

Copper (2.3 µg/L) stimulated acid phosphatase activity at the end of embryonic development in the eggs of the cuttlefish, Sepia officinalis (Lacoue-Labarthe, 2010).

DISCUSSION

This review reveals that various concentrations of copper and mercury at varying lengths of exposure alter the levels of protein, carbohydrate, lipid, phosphatase, and aminotransferase significantly in the different soft tissues and hemolymph of molluscs. Cu and Hg have adverse effects on the biochemical composition and enzymes of molluscs, and their toxicity is metal-, organ-, or species-specific.

The presence of heavy metals is associated with oxidative stress conditions (Semedo et al., 2012) that cause a lowering of the nutritional reserves of molluscs via the degradation of biomolecules like carbohydrates, lipids, and proteins to meet the increased metabolic needs, which get increased above normal levels. Though carbohydrates serve as a readily available energy source during stress, their depletion can be correlated to the increased consumption of carbohydrates because of the anoxia, hypoxia, or anaerobic metabolism caused in order to encounter the heavy metal stress in the environment (Dezwan & Zandee, 1972; Javed & Usmani, 2015). Since after carbohydrate, lipids are utilized to overcome the heavy metal stress conditions, this leads to a reduction in the level of total lipid in the organs of molluscs due to inhibition of lipid synthesis, increased lipase activity, or mobilization of stored lipid by β oxidation (Demir et al., 2005; Jha, 1991). Few studies reported an increase in lipid content, which might be due to increased lipogenesis, inhibition of lipase, diminished lipolysis, diminished transport of lipid, or a shift in carbohydrate and protein metabolism to lipid synthesis (Coley & Jensen, 1973; Swami et al., 1983). The decrease in protein level in the tissues of metal-exposed molluscs might be due to the enhancement of proteolysis, decrease in anabolism of protein, denaturation of protein, or enzyme inhibition (Pottinger et al., 2002; Sastry & Gupta, 1978;

The inhibition of phosphatase activity is related to the breakdown of glycogen to meet the energy demand under stress, a decrease in the transphosphorylation rate, or the uncoupling of oxidative phosphorylation (Kaviraj & Gupta, 2014). The development of osmotic gradients in the lysosome due to the accumulation of toxicants results in the swelling of the lysosome, leading to increased lysosomal lability, which could result in the leakage of CP into the hemolymph, leading to a decrease in CP (Chvapil et al ., 1972; Verity & Reith, 1967). The increase observed in aminotransferase activities might be attributed to a compensatory mechanism attempting to provide energy to drive an impaired metabolism (Knox & Greenguard, 1965). Metals may interfere with substrate binding or bind to the active sites of enzymes, altering enzyme activity

(Ulmer, 1970). Metals are found to inhibit, stimulate, or influence the rate of action of certain enzymes by inactivation, activation, and uncoupling reactions (Suresh & Mohandas, 1990a; Tallendini et al. , 1986).

Lysosomes and the cell membrane are the first targets of xenobiotic, as lysosomes are concerned with the disintegration of foreign bodies and metal detoxification, and the cell membrane functions as the first line of defense against xenobiotic encounters. cid phosphatase is designated as an index of the lysosomal system (De Duve et al ., 1955; Edwards & James, 1987). Lysosomal biomarkers are early warning signals of the biological effects caused by environmental pollutants. minotransferases are useful indicators of metabolic alterations in molluscs (Cheng et al ., 1980). Changes in enzyme activity levels are of great diagnostic value.

The apparent sensitivity of these biochemical and enzymatic parameters’ activities suggests that they have the potential to be promising and reliable biomarkers of water pollution due to copper and mercury and can constitute an important diagnostic tool in toxicological studies. dditionally, the shells of molluscs should be considered a contamination biomarker as they sequester pollutants (Elder & Collins, 1991; Koide et al. , 1982; Protasowicki, 2008). review of the literature also reveals that molluscs can be considered a full-time biomonitor as they react to pollutants and can provide useful information on water quality over time. The “Molluscs Watch” program should be included in environmental surveillance programs to keep watch on the health of the aquatic ecosystem and to protect aquatic biodiversity.

The present review is expected to open up further detailed research in the field of molluscan toxicology, especially on metal-induced pollution aspects in molluscan biochemistry and physiology. It can provide information on the status, trends, and risks facing the aquatic ecosystem. It will be very useful in the fight against pollution by supporting environmental policies, laws, standards, and control methods. It can inform the choice of action to restore ecosystem structures, functions, and services effectively and efficiently in those ecosystems that are already affected by pollution and make important contributions to protecting food resources in aquaculture around the world.

CONCLUSION

Various concentrations of copper and mercury at varying lengths of exposure alter the levels of protein, carbohydrate, lipid, phosphatase, and aminotransferase significantly in the different soft tissues and hemolymph of molluscs. The apparent sensitivity of these biochemical and enzymatic parameters’ activities suggests that they have the potential to be promising and reliable biomarkers of water pollution. Molluscs can be considered full-time biomonitors as they react to pollutants and can provide useful information on water quality over time.

CONFLICT OF INTERESTS

The authors declare that they have no potential conflicts of interest.

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