Potential Domoic Acid (Neurotoxin) Producing Phytoplankton Pseudonitzschia in Indian Coastal Water - Do We Need To Care?

Pseudonitzschia species are potential domoic acid producers, a neurotoxin, responsible for the infamous human HAB intoxication at Prince Edward Island, Canada in 1987, costing human lives. Global warming has widened the reach of these phytoplankton species and it is being reported in Indian waters. We report the occurrence of ten Pseudonitzschia species in the northwestern coastal waters of India, out of which, seven are potential domoic acid producers. The question arises, are we vulnerable to HAB (Domoic Acid) toxicity? In light of the observation that Pseudonitzschia dominates the coastal waters of Veraval and its abundance is increasing with time, the present study briefly synthesizes the available information on the ecology, metabolism, and other relevant knowledge related to the domoic acid production by Pseudonitzschia and assesses the risk of human intoxication through trend and forecast analysis with the possible preventive measures required.


Introduction
About half of the total human population lives within 200 km of coastlines (Creel,2003). According to data on the blue economy by World Bank (Blue economy, 2022),oceanic resources contributeabout $1.5 trillion annually to the world economy, apart from providing much-needed nutritional benefits to human beings. Fish and other edible oceanic natural resources are one ofthe cheap and very good sources of proteins for a large population of coastal communities. According to the data on fish landing by Central Marine Research Institute,India produces3820207tons of fish resources annually (Annual data, 2022) which supports a large coastal community, the health of such aquatic ecosystems are of critical importance in ensuring human health as well as their economic wellbeing. One of the emerging challenges faced by the coastal ecosystems is the outbreak of harmful blooms (HAB) of phytoplankton which sometimes causes the release of toxic secondary metabolites into the water thereby posinga serious threat to the coastal ecosystem,biodiversity and human health. Phytoplankton are microscopic, free-floating and pigment-containingorganisms performing the ecological function of fixing non-bioavailable energy (light energy) into bioavailable chemical form (carbohydrate) much like the plants of terrestrial ecosystems. Apart from the normal function of photosynthesis, a few phytoplankton species produce harmful secondary metabolic products which may be harmful to the ecosystem and/or cause toxicity to other organisms. According to a study by D'Silva et al., 2012, out of the 5000 species of phytoplankton existing in the world's oceans, 7% are reported to form blooms that included diverse phytoplankton groups such as diatoms, dinoflagellates, raphidophytes, prymnesiophytes and silicophytes. Among the bloom-forming phytoplankton, only 2% were reported to be toxic and dinoflagellates contributed 75% to the toxic bloom-forming phytoplankton.39 phytoplankton species are documented as responsible for the formation of algal bloom in Indian waters. In context to Indian waters, the earliest event of toxicity was reported by Hornell in 1908 when massive fish mortality occurred due to an unidentified flagellate bloom. Subsequently, many toxic and nontoxic bloom events have been reported from Indian waters. Appendix 1 summarizes the occurrence of bloom events in Indian waters, their causative phytoplankton species and their effects. Considering the western coastline of India, most bloom events have been reported from Kerala followed by Mangalore and Goa(D'Silva et al., 2012). There has been no report of Pseudonitzschia bloom or its toxicity from any part of Indian water to date. This study reports the first occurrence of Pseudonitzschia (a diatom) bloom along thenorthwestern coastline (Veraval coast, Gujarat) of India. Also, a mini review on bloom events in Indian waters along with the biology of Pseudonitzschia and the eco-physiology of domoic acid production is briefly considered in this paper.Pseudonitzschia produces a neurotoxin called domoic acid and various species of Pseudonitzschiaare well known for causing toxic blooms in many parts of the globe (Lelong et al., 2012). The study assesses the potential risk of toxicity through trend and forecast analysis.

Mini Review-Pseudonitzschia and domoic acid  Toxin-producingPseudonitzschia species
Morphologically,Pseudonitzschia is a diatom with lanceolate or spindle-shaped frustule in its value view. Frustules overlap at value ends to form stepped chains (Tomas 2007 (Lelong et al., 2012).Similar to many other diatoms, Pseudonitzschia often blooms in the upwelling zones where light and nutrient conditions are most favourable. It was observed by Trainer et al. 2008; that Pseudonitzschia blooms are common along the western coast of the continents due to upwelling and water circulation produced by sea floor and coastal topographies. A distribution map documentingthe worldwide occurrence of various species of Pseudonitzschiawas given by Lelong et al., 2012.
 History of bloom events The first incidence of harmful effects produced by Pseudonitzschia was reported from the eastern coast of Prince Edward Island,Canada, in 1987; when many people got ill and three died due to the consumption of intoxicated mussels (Mytilus edulis) (Todd, 1990). Following this, the toxic blooms of P.multiseraewere observed for three years along the eastern coast of Canada (Smith et al., 1990a;Villac et al., 1993). Since then, Pseudonitzschia blooms and production of domoic acid has been observed in many other parts of the world (Shumway 1989;Chang 1993;Hallegraeff 1994;Miguez et al. 1996;Beltran et al. 1997;Lelong et al., 2012).

 Symptoms of domoic acid toxicity
The most unusual and serious symptom of domoic acid poisoning in humans is loss of short-term memory and in some cases, it causes permanent damage to the brain. The poison is not destroyed either by cooking or freezing. Apart from Pseudonitzschia, domoic acid is released by a variety of other macro and microalgae The poison was first discovered from red algae called 'doumoi' (Chondriaarmata) in the Japanese language, in 1958 and was used as a folk medicine in Japan to treat intestinal pinworm infestations (Villac et al., 1993;Mos, 2001).

 General properties of domoic acid
Domoic acid is water soluble and heat-stable amino acid (Hatfield et al., 1995;Leira et al., 1998) but gets degraded by bacterial action (Windust, 1992;Stewart et al., 1998) and by exposure to UV radiation (Wright et al., 1990, Bates et al., 2003. It is also known to chelate iron and thus iron is also considered as a potential degrading agent (Rue and Bruland, 2001). Limitations of nutrients such as phosphorus, silica and metals like iron and copper have been shown to promote toxin production (Pan et al., 1996b;Bates, 1998;Wells et al., 2005). Also, increased levels of salinities, dissolved inorganic carbon and urea have been related tothe enhancement of toxin production

Materials and Methods
Water samples were collected to study phytoplankton diversity from the coastal waters of Veraval, Gujarat, which is a part of the northeastern Arabian Sea. The study was conducted from March 2003 to April 2017.The geographical distribution of all the sampling sites is shown in Figure 1. The relative abundance of phytoplankton cells was calculated to study the extent of dominance of Pseudonitzschia cells over other phytoplankton communities.Decadal trend analysis and forecast analysis for the next 10 years was carried out to understand the pattern of rise and estimate the potential hazardposed by Pseudonitzschia cells.The threshold of bloom initiation and level of risk was determined according to the method proposed by Siegel et al (2002).For this, the mean value for decadal phytoplankton cell counts was calculated and rise in Pseudonitzschia cell counts above 30% of the meanvalue was considered as bloom initiation.

Result and discussion  Reporting
A study on phytoplankton assemblage revealed that diatoms dominated the overall phytoplankton diversity with 77.93% in coastal waters of Gujarat, comprising the northeastern part of the Arabian Sea.
Pseudonitzschiaalone contributed to 26% of overall phytoplankton diversity in the study region.
In order to further understand the effect of seasonal changes in Pseudonitzschia dominance, the study period was temporally classified as fall inter-monsoon, winter monsoon and spring inter-monsoon seasons. Again, diatoms dominated in all the seasons studied, but Pseudonitzschia did not (table 1). Although the abundance of Pseudonitzschia was significantlyhigh in all the seasons, it did not dominate the overall diversity during the two inter-monsoon seasons. Pseudonitzschia dominated with a relative abundance value of 0.26,exclusively in the winter monsoon season.As mentioned in the mini-review above, Pseudonitzschia grows best in upwelling zones and coastal waters where nutrient conditions are favourable for their growth and multiplication.During winter monsoon, cooler water at the surface sinks towards the bottom causing upwelling (Motwani et al., 2014). This upwelling water brings along nutrients from the bottom, turning the nutrient conditionfavourablefor the growth of Pseudonitzschia. Whereas in fall and spring inter-monsoon seasons, the hydrological condition is reversed and the water is stratified. Nutrient supply in stratified water is low thus lowering the overall phytoplankton diversity and thereby abundance of Pseudonitzschia as well. there was no presence of toxin. The possibilities may be either these Pseudonitzschiaspecies were not producing toxins or the produced toxin wastoo low to produce any harmful effect and that they were assimilated into the aquatic system.
The decadal trend of Pseudonitzschia abundance showed that its abundance in the coastal waters of Gujarat was almost zero ( Figure 3) till 2004. Later, after its introduction in the region, itincreased in number continuously such that it can now be considered an invasive species. In 2015, its cell countoutcompeted the overall phytoplankton diversity and formed a bloom due to the occurrence of two extremely severe cyclonesChapala and Meghone after the other in October-November.  Recalculated results also showed an increasing trend forPseudonitzschiacell counts. Forecast analysis for the next 10 years showed that Pseudonitzschia is likelyto increase( figure 4) and therefore is a potential future hazard for the region. Veraval is a known fishing centerand a busy shipping port.Apart from fishing and shippingactivities, the shipbuilding industries of Veraval also influence the water chemistry of the region. A lot of organic matter and metal wastes are disposedinto the adjacent coastal waters.The presence of Somnath temple (an old Shiva temple of historical importance)makes Veraval an important pilgrimage and tourist center of the state. Commercial activities make Veraval prone to excessive eutrophication and pollution, causing an imbalance in the proportion of essential nutrients such as nitrogen, phosphorous and potassium. Such eutrophic conditionsseemingly turnedfavourable for the growth of Pseudonitzschiaand can act as a potential trigger for the release of domoic acid in future. According to Bhat and Matondkar, 2004, pollution and nutrient enrichment due to anthropogenic activities are the major factors that trigger and stimulate the growth of bloom-forming species.Both these triggering agents prevail in Veraval and undoubtedly are responsible for the continuously increasing abundance of Pseudonitzschia.

 Risk Assessment
Results of the threshold method showed that Pseudonitzschiacells were higher than 50% of the mean value for overall phytoplankton diversity. It not only formed bloom but also posed a major risk tothe human health and fishing industry of the region under concern. The dominance of Pseudonitzschia and its increasing trend is definitely an alarming situation. To understand the potential risk due to the presence of Pseudonitzschia in alarmingly high numbers, the risk assessment method proposed by the government of South Australia, Department of Education and Children'sServices was followed. The outcome of the risk assessment process showed that human health, ecosystem and fisheries industries are extremely vulnerable to possible catastrophic effectsdue toPseudonitzschiabloom in coastal waters off Gujarat. Further ahead, there is no awareness or management strategiesundertaken to handle the possible risk (table 2). Although, there were no reports of any harm by toxin release in the study area. There is anextreme risk of domoic acid production in the near future and by all means it can affect the commercial activities of the region as well as the health and well-being of the human population that consumes intoxicated fishes.

 Need for research
The absence of toxication reports does not ensure that there is no production ofneurotoxins in the region. There may be neurotoxin production but in very low concentrations that it does not cause any mortality or it may be due to lack of awareness and research carried out in the region that domoic acid remains unnoticed. Detailed studies on the presence of toxic Pseudonitzschiain Indian waters and their toxin production activities are required. The toxin-producing stimulus to these species and highly sensitive protocols to detect the presence of toxins at very low concentrations are also subjects of research required in this region.

 Management approaches
Occurrence of toxin-producingPseudonitzschiaspecies in significant proportion over other phytoplankton types call upon a need for continuous monitoring of Pseudonitzschia abundance, water quality, nutrient levels and anthropogenic factors that can trigger the release of domoic acid. Such monitoring can help to protect the consumers and the seafood industry. Also, fishermen and people inhabiting the coastal regions of Gujarat need to be educated about the potential hazard. Eutrophication is one of the causes of Pseudonitzschia dominance in the region. Sustainable approaches to control industries and shipping activities need to be adopted to curb the approaching hazard.

Conclusion
The coastal waters of Veraval are dominated by a potential neurotoxin-producing phytoplankton 'Pseudonitzschia'. The abundance of Pseudonitzschia cells showed an increasing trend over a decadal time period. Increasing abundance is not only disturbing the present diversity but also posing a potential risk of being detrimental to human health, the ecosystemand the fisheries industry of the state. Presently there is no awareness or report of the possible threat. There is indeed a need for awareness, research and management activities to handle the fore coming threat before it actually takes a toll on human health and finances.

Acknowledgement
The work was funded byDST-SERB, Gov. of India, under the NPDF scheme. We are grateful to Dr. Ashish Jha and Sreejith Thilakan from Central Institute of Fisheries Technologies, Veraval and the