A Commentary on the Use of Bivalve Mollusks in Monitoring Metal Pollution Levels

The objective of this commentary is to promote the use of bivalves as biomonitors, which is a part of the continual efforts of the International Mussel Watch. This commentary is an additional discussion on “Bivalve mollusks in metal pollution studies: From bioaccumulation to biomonitoring” by Zuykov et al., published in Chemosphere 93, 201–208. The present discussion can serve as a platform for further insights to provide new thoughts and novel ideas on how to make better use of bivalves in biomonitoring studies. The certainty of better and more extensive applications of mollusks in environmental monitoring in the future is almost confirmed but more studies are urgently needed. With all the reported studies using bivalves as biomonitors of heavy metal pollution, the effectiveness of using Mussel Watch is beyond any reasonable doubts. The challenge is the development of more accurate methodologies for of heavy metal data interpretation, and the precision of the biomonitoring studies using bivalves as biomonitors, whether in coastal or freshwater ecosystems. Lastly, inclusion of human health risk assessment of heavy metals in commercial bivalves would make the research papers of high public interest.


Introduction
Firstly, the well-written review paper published by Zuykov et al. [1] promoting the use of bivalves as biomonitors is focused on and discussed in the present commentary paper. As of January 2021, this paper had been cited by 165 papers based on Google Scholar. This monitoring paper using bivalves is a part of a continual effort of the International Mussel Watch. This mussel monitoring work should have been highly commended and supported from its inception when it was suggested by Goldberg [2] and should continue to be for educational purposes. This is because of the fact that the pollutant levels in bivalves will greatly affect human health.
We must say that it is not our intention to offend the good researchers in the paper by Zuykov et al. [1], but our honest comments are based on science. As researchers working on biomonitoring studies using mussels since 1998, we want to humbly comment and add more discussion on the above review paper, based on the following highlighted points of view as presented the paper. 1.
The uses of bivalves as biomonitors of metal pollution are primitive methods.

2.
The use of shells to construct pollution history blueprint is futile.

3.
There has not been any documented evidence of severe health effects of bivalves due to metal accumulation.

Comments and Discussions
Two of the specific goals of the paper by Zuykov et al. [1] were to (1) "discuss the biomonitoring of metal pollution using bivalves", soft tissues and shells as a mean of environmental "health monitoring", and (2) "reveal additional information of metal pollution in aquatic environments based on the observation of the internal shell surface through scanning electron microscope". However, these objectives were not answered and not actually summarized or briefly discussed in the abstract.

The Uses of Bivalves as Biomonitors of Metal Pollution Are Primitive Methods
It seems to the general readers that the biomonitoring of metals using bivalves is not effective and is an old research method. We disagree with this highlighted point because there has been an influx of reported studies, before, now and expectedly in the future too, using bivalves for pollution studies. As reviewed in Table 1, many such studies can be found in different countries up until January 2021. In our view, we would not say "primitive" since this could totally discourage the use of bivalves in metal pollution monitoring and funding for such studies would not be continued. We think, besides bioaccumulation data, the effectiveness of using identified bivalves for the biomonitoring of metal pollution for spatial distribution and comparison should be improved upon and the accuracy enhanced, in future studies.
In fact, Zuykov et al. [1] made a substantial, careful and excellent review on the use of bivalve soft tissues and shells in biomonitoring studies from numerous papers. Even similar early papers by Boening [3], Yap et al. [4] and Yap [5] have given suggestions for biomonitoring assays using marine mussels. This trend continues in recent papers such as Nędzarek et al. [6] and Wepener and Degger [7]. All of this clearly shows the potential of mollusks as sentinel organisms of heavy metal pollution for future studies. In particular, Yap [5] and Yap et al. [8] proposed that the recommended criteria for marine mussels could be applied to other mollusk species, establishing them as good biomonitors of heavy metal pollution. None of these papers mentioned that "the biomonitoring work is not far advanced". Therefore, in our opinion, the uses of bivalves as biomonitors of metal pollution are far reaching methods instead of being primitive. The paper entitled "From bioaccumulation to biomonitoring" is somewhat confusing to the readers too. However, when the abstract is carefully looked into, "biomonitoring" means "estimation of environmental quality". In fact, biomonitoring is better explained as "regular and systematic use of living organisms to evaluate changes in environmental or water quality that involves repetitive measurements of pollutants/chemicals", which is central to aquatic toxicology and ecotoxicology. In the science of analytical chemistry, biomonitoring is the estimation of the body burden of poisonous synthetic compounds, elements, or their metabolites in bioorganic substances [98].
Before we can better estimate the environmental quality, we think the current challenges now are to enhance the precision of the biomonitoring studies using bivalves as biomonitors, whether in coastal or freshwater ecosystems. These challenges have been little discussed in the recent literature ( Table 1). The metal bioaccumulation data in bivalves could be influenced by several abiotic and biotic factors. For example, the abiotic (such as pH, salinity, etc.) and the biotic (sizes, gender, genetic differentiation, predation, competition) factors could simply alter the metal bioaccumulation data in the body tissues of the bivalves [99].
Based on the title of the paper again, most ecotoxicologists will understand that the use of bivalves for biomonitoring purposes is of high novelty. However, bivalves have been widely employed and recognized as good biomonitors of four major collective pollutants-namely, halogenated hydrocarbons, transuranics, heavy metals and petroleum, as mentioned in the Mussel Watch Program [2]. This is because mussels have many of the important characteristics of good biomonitors [100,101] of coastal pollution. According to Farrington et al. [101], bivalves are widely distributed in coastal waters across the globe, are sessile in nature, are capable of accumulating pollutants at high concentrations (by factors of 10 3 to 10 5 ), seem to be unaffected by pollutants, are important seafood products heavily consumed in certain parts of the world and therefore can be a risk to human health. For Perna viridis particularly, extensive studies had been reported from the Asia-Pacific coastal regions [102,103]. From at least 65 of the high impact publications as seen in Table 1, four patterns can be deduced.
Firstly, publications on metal studies in mussels have been consistently and widely accepted worldwide in more than 50 countries around the world's coastal regions, as summarized in Table 1, spanning from the 1970s until January 2021.
Secondly, there have been continuous efforts made and research grant allocations given to study heavy metal levels in bivalve mollusks in both marine and freshwater environments, but mainly in marine mussels, ever since the introduction of the famous International Mussel Watch, initially proposed by Prof. Goldberg [2]. This shows that metal pollution studies using mussels are not only truly scientific research studies based on biomonitoring, but they have also triggered other newly emerging scientific studies and topics.
Thirdly, the summary in Table 1 highlights that biomonitoring studies using mussels occur in developed, developing and underdeveloped countries all around the world. This greatly signifies that Mussel Watch is a cost-effective study and the idea has spread in a very positive and fruitful manner in all coastal areas around the world. The benefits include developing expertise in the areas of ecotoxicology, biology and environmental sciences, which eventually lead to opportunities for training postgraduate research students.
Fourthly, the common heavy metals such as Hg, Cd, Cu, Pb and Zn have always been focused upon with Ag, Al, As, Ba, Ni, Co, Cr, Fe, Mn, Se, Sr, Sn, Ti and V. In fact, the biomonitoring studies using bivalves have been expanded to more metals or elements including rare earth elements (Table 1). Therefore, if biomonitoring studies using mussels are "not far advanced", then this work is a futuristic study that is influential in many regions around the world with its ever-expanding trends, although it is traditional in its origins and ideas.

The Use of Shells to Construct Pollution History Blueprint Is Futile
We have conducted a comprehensive review covering papers published up until January 2021 on the use of bivalves' shells for the biomonitoring of metals. In addition, based on the review presented in Table 2, more related studies give evidence that such studies are continuing now and in the future. The main reason for this is because shells have potential for biomonitoring metal pollution, and it is believed that they can be reconstructed to reflect the metal pollution history. This challenge remains. This study suggested that shells of bivalves may be an essential and underutilized assessment tool for pollutant assessments in the environment (3 sites near Pangnirtung, Northwest Territories; 1985).
[108] The findings based on stepwise regression analysis showed that the transport of Cd, Pb and Zn into the mussel shells could have caused the shell deformities.

Perna viridis Cd and Pb
Suggested the potential of periostracum of P. viridis as a biomonitor for Pb but not for Cd. However, further studies should be conducted to prove the potential of periostracum as a good biomonitoring tissue for heavy metal pollution in tropical coastal waters. [125] 27

Perna viridis Cu and Zn
Periostracum is suggested as a good biomonitoring tissue for Cu, but not for Zn, based on the higher levels of Cu in periostracum in comparison to soft tissues and closer relationship of the Cu between periostracum and sediment.
[103] Enriched metals (Fe and Mn) were found in bivalve shells from hydrothermal fields with black smokers. It was also evident that in the early ontogeny of the shells essential metals such as Fe, Mn, Ni, and Cu were more actively accumulated. The shells of the bivalve displayed efficient accumulation functions due to high concentration factors of majority of the metals (seven hydrothermal vent fields of the Mid-Atlantic Ridge). [126] 29

Mytilus galloprovincialis
K, Ca, Fe, Cu, Zn, Si, Sr, Al, Mn, Pb, As, Hg, V, Cr, Sn, Cd, Ni and Co The study showed higher metal levels in the soft tissues in comparison to shells but shells might also give relevant information on the environmental metal pollution status. Two visible patterns of bioaccumulation in soft tissues (As, Cd, Hg, Pb and Sn) and shells (Co, Cr, Mn, Ni, Pb and V) were also found, indicating strong associations, most likely of anthropogenic origin (Cape Town Harbour, South Africa, 2011). [60] 30

Perna viridis
Ag, As, Be, Co, Cr, Cs, Hg, Li, Mn, Se, Sr and V It is difficult to explain the outcome of this study as all metal data on soft tissues and shells presented were after the transplantation periods from a polluted site to two unpolluted sites in the Straits of Johore. [127]

31
Unio tumidus Zn, Cu, Fe, Pb, Ni and Cd These results reflected contemporary anthropogenic pollution of the environment with heavy metals and confirm the possibility of using the shells in the assessment of heavy metal pollution levels (Szczecin Lagoon, SW Baltic basin). The main cause of malformations in green mussels was suspected to be Pb, Hg and Sn (Jakarta Bay, Indonesia). [132] The original idea of using the shell as a reliable biomonitoring material for the reconstruction of pollution history (see [133][134][135][136][137][138]) is acceptable and is believed to be workable and reliable when compared to the use of soft tissues. This is the main reason why so many researchers conducted such related studies using molluscs' shells for the biomonitoring of metal pollution, to compare the current with the past history of metal bioaccumulation in bivalve shells as reviewed in Table 3. The reason why we compared the previously collected shells with the current ones is because we can logically establish the past pollution history as shells can be easily stored without having to be kept in a freezer (Table 3).
Overall, researchers are trying to provide more data and evidence, but the reconstruction of pollution history using shells remains a big challenge. Therefore, while we wait for more evidence to prove the positive usage of mollusc shells for elucidating pollution history, we think it is premature to say "Shells cannot be reliably used for the reconstruction of the pollution history". In fact, almost all the papers reviewed by Zuykov et al. [1] concluded on the positive use of bivalves' shells for biomonitoring of metal pollution and none stated the contrary. Rather, we think more studies are needed to establish the use of bivalves' shells as good biomonitors and to reconstruct the pollution history of heavy metals.

There Has Not Been Any Documented Evidence of Severe Health Effects of Bivalves Due to Metal Accumulation
If the paper is only based on the reviewed literature, we disagree with the statement that "There are no effects of high metal bioaccumulation on the health status of bivalves". In fact, a number of biomarkers have been used to show the health effects due to metal bioaccumulation in the mussels and these have also been evidently proven (Table 2). For example, the condition index (CI) of bivalves has been widely used as a health status measure in response to high metal bioaccumulation in bivalves due to pollution effects [139]. Unless there are other major factors, the metal levels in the body tissues and the CI of bivalves are negatively correlated. Based on their studies, de los Ríos et al. [140] reported the detrimental effects on mussels' health from metals and xenoestrogenic endocrine disruptors found in some discharges. Signa et al. [119] reported that a lower condition index and phospholipids, as well as higher total and neutral lipids in mussels from Augusta, indicated the occurrence of physiological and biochemical stress responses to metal exposure and accumulation. Table 3. A review on the use of bivalves' biomarkers in metal pollution studies on bivalves from some of the available literature.  Integrated biomarker response (metallothioneins, glutathione S-transferase, catalase, acetylcholinesterase and RNA:DNA ratio).

Mytilus galloprovincialis
Cu, Ni, Pb, Cr, Cd, Fe and Zn Intralysosomal metal levels in digestive cells, metallothionein content in digestive gland tissue, peroxisome proliferation, the exposure component of the bell-shaped changes in digestive gland AOX activity, intracellular accumulation of neutral lipids in digestive gland diverticula; ALP level in mantle (gonad) of male mussels. Genotoxicity biomarkers: MN frequency measured in haemocytes. Oxidative stress biomarkers: MDA levels in digestive gland and LPF accumulation in digestive cells. General stress biomarkers: Lysosomal membrane labilisation period in digestive cells; cell-type composition of digestive tubule epithelium. Population fitness biomarker: accumulated mortality in air exposed mussels against exposure time (days) and LT50 (days). [144] 8 Mytilus sp. As, Cd, Co, Cr, Cu, Hg, Methyl-Hg, Mn, Ni, Pb and Zn Haemocyte lysosomal stability, frequency of irregular nuclei in haemocytes, and frequency of micronuclei in haemocytes [145] 9.

Mytilus galloprovincialis
Cd, Cr, Cu, Fe, Ni, Pb and Zn Lysosomal membrane stability and lysosomal structural changes and changes in cell-type composition in digestive gland epithelium [148] 12

Mytilus galloprovincialis
Hg, Cd, Pb, Cu, Zn and As Antioxidant enzymatic activities, lipid peroxidation, and the physiological rates integrated in the scope for growth biomarker (clearance rate), biological variables (shell thickness), condition index, gill index, gonado somatic index, hepato somatic index, total reproductive potential, sexual maturity index.

Mytilus galloprovincialis
Cu, Cd and Hg Oxidative-damage of protein expression and modification-damage on the protein synthesis machine integrity and specifically on translation factors and ribosomal proteins expression and modifications.
[163] Mussel gills in metal pollution biomonitoring is a promising tool for the detection of changes in bioavailable metals in the environment, especially for essential metals such as Cu and Zn. [174] Riveros et al. [141] measured cellular biomarkers (the levels of vacuole formation and the amount of lipofuscin granules in the digestive system as well as lysosomal stability in haemocytes) in the mussel Perumytilus purpuratus from the intertidal zones of San Jorge Bay, Antofagasta, Chile. Al-Subiai et al. [143] concluded based on the multibiomarker approach using Mytilus edulis that a visible relationship between genotoxicity and higherlevel effects was present and this could be used to determine various short-and longterm toxic effects of Cu. Marigomez et al. [144] reported that biomarkers in depicted site-specific profiles served as essential diagnostic tools for health assessments of metal pollution not just of mussels but of the marine ecosystem as well. Their study supported a combined use of both caged and native mussels in highly polluted areas to monitor the biological effects of pollution in mussels through the integrative biomarker approach. Brooks et al. [150] highlighted the biological effects of pollutants on the blue mussel (Mytilus spp.) to evaluate the effluents discharged from the Sydvaranger mines. The results of the integrated biological responses were in line with the source of pollution judging by the distance and location between the mussels and the discharge outlet and the expected exposure to the mine effluents. González-Fernández et al. [151] concluded that the vast variability of the population of the mussel, Mytilus galloprovincialis, caused by environmental factors such as food availability in a certain monitoring program might conceal the effects of pollutants on the biomarkers. Chandurvelan et al. [152] reported that the specific tissue or the whole organism response of Perna canaliculus towards metal pollutants reveals crucial information on the biological stress responses, denoting the importance of such measurements in biomonitoring programs in New Zealand. Giarratano et al. [147] reported that the gill of the mussel Aulacomya atra is an actively proliferating tissue and is the first target of contaminants (Fe, Al, Zn, Cu, Cd and Pb) present in the water, so that changes in its antioxidant system can provide an earlier warning signal than changes in other tissues. Lekube et al. [148] concluded that cellular biomarkers in Mytilus galloprovincialis were extremely sensitive and quick to respond to changes in environmental pollutants such as heavy metals. The results obtained by Tsangaris et al. [62] confirmed the usefulness of the integration of biological effect measurements and chemical analysis in Mytilus galloprovincialis for the assessment of chemical contamination including those by Cu, Ni, Fe and Zn in coastal waters. Other studies using biomarkers as evidence to show the effects of accumulated metals in relation to the health of the mussels investigated are shown in Table 3.
According to Lam and Gray [175], biomarkers can be used for the quantitative determinations of physiological and biological changes that are observed in cells, body fluids, tissues or organs of an organism. These serve as indicators of exposure to xenobiotics and their effects. Therefore, the accumulated metals on the health of bivalves can be reflected by looking into the biomarkers of the mussels. Table 4 shows a review on the human health risk assessments of heavy metals in bivalves from some available literature. With the increasing trends of anthropogenic inputs into aquatic ecosystems, the commercial shellfish from these areas are of much public concern. Therefore, from Mussel Watch monitoring to human health risk assessment of heavy metals are of high significance [176,177]. It seems that with heavy metal data in the bivalves without the human health risk assessment of the heavy metals would make the whole research finding of low novelty. 2

Cristaria plicata
Zn, Pb, Cd, As, Cu and Cr; the hazard index (HI) values for adults and juveniles were higher than 1, suggesting significant risks of noncarcinogenic effects to humans by exposure to multiple metals.

Mytilus galloprovincialis
Pb, Cd, Cr, Ni, Co, Cu, Zn, Mn and Fe; the Cr measured in mussels was considered "extreme", according to the consumption rate limit for mussels which limits their consumption to 0.5 kg/day.  11

Brachidontes rodriguezii
Cd, Cu, Pb, Mn and Fe; the metal contents in mussels met the national and international standards for safe consumption.

Perna viridis
Cd, Cu, Fe, Pb Ni and Zn. THQ values were <1.0 for average level mussel consumers but higher than 1 for high level mussel consumers in some sites.

Conclusions
With all the reported studies using bivalves as biomonitors of heavy metal pollution, the effectiveness of using Mussel Watch is beyond any reasonable doubts. The challenge is on the development of more accurate methodology of heavy metal data interpretation and the precision of the biomonitoring studies using bivalves as biomonitors, whether in coastal or freshwater ecosystems. Such ideas have been marginally proposed in the literature [189,190]. In addition, human health risk assessment of heavy metals in commercial bivalves will be of much public interest. Therefore, inclusion of consumer perspectives on the heavy metal data is of high importance. Lastly, the Mussel Watch approach could be proposed as Crop Watch in studies of ecotoxicological genetics [191].
This commentary is an additional discussion on "Bivalve mollusks in metal pollution studies: From bioaccumulation to biomonitoring" by Zuykov et al. [1]. It is hoped that this communication paper will serve as a platform for further discussion that can provide new thoughts and novel ideas on how to make better use of bivalves, both marine and freshwater, in biomonitoring studies. It is certain that more future studies using bivalve mollusks as biomonitors of pollution are much needed.