Spatiotemporal Distribution of Harmful Algal Flora in the Tropical Estuarine Complex of Goa, India

Mandovi and Zuari estuarine complex is monsoon-influenced estuaries located along the central west coast of India. During the past few years, there has been an increase in nutrient loading specially during monsoonal runoff which is responsible for the growth of harmful algal flora. To understand occurrence and distribution of harmful algal blooms species, daily/alternate day samplings were carried out in Mandovi and Zuari estuaries during 2007-2008 and 2008-2009 periods, respectively, comprising of monsoon (June–November) and nonmonsoon (December–May). In Mandovi, total 54 HAB species with 49 in monsoon and 36 during nonmonsoon period were reported. In Zuari, total 46 HAB species with 38 in monsoon and 41 were reported during nonmonsoon period. Bray-Curtis cluster analysis based on log-transformed phytoplankton density detected seven well-defined groups revealing spatiotemporal variability. The density of the dominant harmful algal species was significantly positively correlated with nutrients, but negatively correlated with salinity. The results of the study indicate that monsoon plays an important role in occurrence and distribution of harmful algal species having direct correlation with salinity variations and nutrient loading.


Introduction
Harmful algal blooms occur when the algal cells in the marine or fresh water grow out of proportion causing economic loss and severe impacts on marine life and human health [1]. The global study reveals that HAB-causing species are potentially harmful due to their physical structure, for example, several species of genus Chaetoceros, Ceratium, and Prorocentrum [2,3]. Some are potentially toxic even at low concentrations (few cells per litre) and produce toxic effects affecting the marine life whose intern affects the human health.
In India, "red-tide" events were caused by Noctiluca miliaris along southern Kerala coast [4][5][6], and severe fish mortality was observed. Paralytic shellfish poisoning outbreak was observed in Mangalore along the west coast where the causative organism was not known [7,8], and the stench of Cochlodinium polykrikoides was observed along the southern Malabar Coast by [9]. Many cysts of toxic dinoflagellate species have been reported in the sediments along the south west of India during the southwest monsoon period [10,11]. "Red tide" event was caused by Coscinodiscus centralis on south west coast of India [12].
The occurrences of HAB species have been linked to a number of factors such as impact of weather conditions on water parameters like salinity, temperature, currents, nutrient concentrations, monsoonal pattern, and also the geomorphology of the location [13,14]. Combination of some of these factors provides optimal conditions for HAB species to transform into bloom.
The Mandovi and Zuari are the most important estuaries of Goa along west coast of India and are typical tropical, tide-driven estuaries [15]. The estuaries differ in their geomorphology and rainfall pattern. The Mandovi estuary has wide mouth region and longer flushing period compared to Zuari [16]. The major source of pollution in the Zuari is the Mormugao port situated at Mormugao bay. In recent years, the anthropogenic activities like construction of jetties, ship building and boat traffic (cruising), mining activities, sewage discharge, agricultural runoff, and industrial effluents have increased in these estuaries [17][18][19][20][21]. This has lead to  the increase in the macro-and micro-nutrients concentration specially nitrate and phosphate, suspended particulate matter, and trace elements influencing the algal blooms. Reports of harmful algal blooms have also been reported from Goa coast [22,23]. However, no intensive study has been carried out on the HAB-forming species from these regions. Therefore the aim of the present investigation was (1) to study the spatiotemporal distribution of harmful algal species with respect to their occurrence and abundance and (2) the important environmental parameters that control these variations in the Mandovi and Zuari estuaries. upstream from the mouth of the estuary, and in Zuari, the site (St.2) was ∼13 km upstream from the mouth of the estuary (Figure 1). These sites were chosen for two major reasons: first, because of its accessibility from the National Institute of Oceanography (NIO), vessel CRV Sagar Shakti which was to be anchored at the point during the entire southwest monsoon season, and second, on account of the large salinity range (0 to 37 psu) that this locations experienced [16]. At the highest high tide, the maximum depth at the sites was 6 m and 9.5 m in Mandovi and Zuari estuaries, respectively. Water samples were collected using a Niskin sampler attached to the Sea Bird CTD. The samples were then immediately transported under cold and dark conditions for further processing at the National Institute of Oceanography Laboratory.

Physicochemical Parameters.
Rainfall over the entire west coast of India was obtained from the India Meteorological Department [24]. Salinity was measured with a "Salinometer" (Atago S/Mill, Japan, Salinity range 0∼ 100 psu, resolution 1 psu between 10 and 20 • C).
The Scientific World Journal 3 2.4. Taxonomy of HAB Species. Samples for HAB species taxonomy and cell counts were collected in 500 mL opaque plastic bottles, fixed with a few drops of Lugol's iodine, preserved in 3% buffered formaldehyde, and stored under dark and cool conditions till further analysis. Samples were concentrated to 5-10 mL by carefully siphoning the top layer with a tube covered with a 10 µm Nytex filter on one end. Sample concentrates were then carefully transferred to a 1 mL capacity Sedgwick-Rafter and counted. Phytoplankton cell identifications were based on standard taxonomic keys [26][27][28], and cell counts were carried out in duplicate.

Statistical Data Analysis.
Data processing was done using PRIMER version 5.2.8 [29]. Bray-Curtis similarity index was constructed based on HAB density after log transformation. Following the division into groups from results of cluster analysis, the species having the greatest contribution to this division were determined using similarity percentage program (SIMPER). Principal component analysis was carried out using statistical package version 6.0.
(StatSoft, Oklahoma USA). The purpose of the analysis is to see the effect of environmental variables on the abundance of dominant HABs species. The first principal component factor 1 accounts greater variability, and each succeeding factor explains the remaining variability possible in the data set. The ordination results for the first two most important factors (factor 1 and factor 2) were retained. Pearson's correlation was carried out in order to show the strength of association between the HAB species and environmental parameters.

Salinity.
In Mandovi estuary, average salinity varied between 5 and 29 psu during monsoon with lowest in the month of July. The average salinity varied from 30 to 33 psu during nonmonsoon period (Figure 3(a)). During monsoon period in Zuari estuary, average salinity varied between 6 and 29 psu, and lowest salinity was recorded in August. In nonmonsoon period, average salinity varied between 27 and 31 psu (Figure 3(b)).

Spatiotemporal Distribution of HAB Species in
Both the Estuaries. The list of phytoplankton species which are potentially harmful and toxic with their cell abundance in both the estuaries is given in Table 1. In Mandovi, total 54 HAB species with 49 in monsoon and 36 during nonmonsoon period were reported. In Zuari, total 46 HAB species with 38 in monsoon and 41 were reported during nonmonsoon period. The 54 HAB species in Mandovi comprised of 33 potentially harmful and 21 potentially toxic species. In Zuari, 46 total HAB species consisted of 26 potentially harmful and 20 potentially toxic species. The Bray-Curtis cluster analysis based on the log-transformed phytoplankton density detected seven well-defined groups revealing the spatiotemporal variability ( Figure 4). Group I consisted of the monsoon samples of Mandovi estuary, while groups II (December-March) and III (April-May) consisted of the nonmonsoon months of Mandovi. Group IV consisted of the peak monsoon months (June-July) of Zuari, while group V consisted of the months of August-October, January. The nonmonsoon period was clustered into two groups, that is, group VI (November-December) and group VII (February-May). SIMPER analyses detected the species   that contributed to the grouping and are summarized in Table 2.

Discussion
The present work provides a detailed study of the harmful algal bloom-forming species in two tropical estuaries, that is, Mandovi and Zuari along the central west coast of India. These estuarine systems receive large amount of runoff from June to September (south west monsoon); as a result, they are fresh water dominated during monsoon. Though partially landlocked, they are exposed to constant flushing and flooding, which considerably affect the environmental features of the estuaries. The study reveals the fact that daily/alternate day observations give more detailed account on the day-to-day changes in the phytoplankton biomass pattern especially on spatiotemporal distribution of harmful algal species with respect to the environmental parameters. Among the nutrients, nitrate was highest during monsoon in the Mandovi estuary, whereas silicate was high in the Zuari estuary throughout the study period. In comparison with earlier studies, an increase in the nutrient input [17][18][19][20][30][31][32][33][34] is reported. The Mandovi-Zuari estuarine complex is the main transport route of the iron ore to the Mormugao harbour. In Mandovi, per year 5.21×10 6 m 3 of the sewage is disposed [35]. Land-based runoff was over 6004 Mm 3 [36] during the monsoon wherein nutrients get transported to the estuaries leading to loading of the nutrients which is a major factor responsible for the growth of the HAB species [37][38][39][40][41][42][43].
The HAB species showed seasonality in the distribution as reflected in the Bray-Curtis similarity index which was purely based on the abundance of HAB species during each month. Thalassiosira rotula, Rhizosolenia fragilissima, and Coscinodiscus concinnus showed temporal variation and The Scientific World Journal was positively related with nitrite and rainfall. Coscinodiscus concinnus was reported in Mahanadi estuary during postmonsoon and premonsoon periods due to their eurythermal and euryhaline nature and is known to grow quickly in estuarine conditions [44]. Rhizosolenia delicatula dominated in Mandovi estuary during the monsoon period. However, this species may occur anytime regardless of the monsoon season, but with varying cell densities.
In the present investigation, potentially harmful pinnate diatom Cylindrotheca closterium was observed with high  abundance in more saline waters during the nonmonsoon period in both the estuaries. The abundance was highest in Mandovi estuary during April-May (Table 2). Earlier this species was reported with high abundance in the Zuari estuary [45]. Coscinodiscus centralis, Coscinodiscus wailesii, Protoperidinium brevipes, Protoperidinium steinii, Distephanus speculum, Ditylum brightwellii, Dinophysis caudata, Gymnodinium splendens, and Prorocentrum micans showed strong positive relation with rainfall, nitrate, phosphate, and silicate (Figure 3(a)). These species are brought into the estuary during sea water influx from coastal water and are known to have preference for phosphate and silicate [40,42]. During monsoon period, estuaries are rich in inorganic phosphate. In Zuari estuary, the source of phosphate is largely from the neritic waters, while in Mandovi, the high input is from the large number of tributaries [46]. Especially dinoflagellates like Protoperidinium brevipes, Protoperidinium steinii, Dinophysis caudata, Gymnodinium splendens, and Prorocentrum micans have diverse habitat and have ability to swim and sink under nutrient-stressed conditions [47,48]. Gymnodinium splendens was reported for the first time during the monsoon phase in Mandovi. This species is known to proliferate in nutrient-enriched waters and also possesses the nutrient retrieval strategy: diel migration of nutrient-depleted dinoflagellate into nutrientrich layers [49]. In general, dinoflagellates are known to grow best in environments that are rich not only in nitrogenous nutrients, but also in humic acid, fulvic acid, and other dissolved organic compounds that constitute the bulk of the colored dissolved organic matter (CDOM) pool [50][51][52].
One important source of CDOM is litter from the mangroveladen banks upstream of the Mandovi River. During the monsoon, constant flooding and flushing of the mangrove beds causes leaching of CDOM from decaying litter. The bloom of Protoperidinium has been reported off Mangalore [53]. Silicoflagellate Distephanus speculum was observed but with low abundance and reported to cause depletion in oxygen concentration and produces harmful red tide [54].
The bloom of potentially harmful species Ditylum brightwellii observed in Zuari estuary during the monsoon The Scientific World Journal 9 period is a noteworthy as this species is known to occur in lower abundance in this estuary in other seasons [45]. The occurrence of Leptocylindrus danicus, Leptocylindrus minimus, Rhizosolenia setigera, Rhizosolenia stolterforthii, Gymnodinium breve, Prorocentrum gracile, Scrippsiella trochoidea, Chaetoceros curvisetum, and Pseudo-nitzschia seriata during the monsoon implies that they are typical monsoonal species which prefer low saline conditions. Gymnodinium breve, another toxic species, which showed spatial distribution with high abundance during monsoon period is of concern. This species can withstand shear/stress effects with autoregulated behavior which allows faster growth at lower light levels in waters enriched with nutrients [47,49]. Prorocentrum gracile and Scrippsiella trochoidea were reported to grow in chemically disturbed (nutrient enriched through anthropogenic activities) marine waters [49]. Domoic acidproducing species of Pseudo-nitzschia seriata and Pseudonitzschia multiseriata have shown spatiotemporal variation in both the estuaries specially during the nonmonsoonal period when the nutrients are elevated but declining. Cultural experiments on these species showed that deficiency in silicate and phosphate triggers more domoic acid production [55].
Ceratium furca that showed spatiotemporal variability with high abundance during monsoon in Zuari estuary has the ability to take additional nutrient sources by mixotrophic feeding which might contribute to bloom formation and persistence allowing population growth even in low-nutrient conditions [56]. Potentially harmful species of Ceratium furca and Skeletonema costatum are found to be preponderant of these estuaries, euryhaline type, and grows in nutrient-enriched waters [45]. Skeletonema costatum is a cosmopolitan species found to grow at a wide range of salinity with high silicate concentration [45]. In this work, high abundance of this species was found in the Zuari estuary during the monsoon period. Bloom of Skeletonema costatum is also reported as indicator of pollution in the marine waters [44,57]. Ammonia also plays important role in the growth of HAB species [38,40]. Recent studies in Mandovi reported 1.79 µmol of ammonia during the monsoon period [20]. Low salinity and high nutrients during the monsoon period favour the growth of HAB species in both the estuaries.

Conclusion
It can be concluded that monsoon acts as a major player in the spatiotemporal distribution of large number of harmful algal species. Abundance of HAB species is related to the increase in the nutrients in both the estuaries. The high level of nutrients is a result of increased anthropogenic activities in the two estuaries. Since fishery is an important activity in this region and during monsoon season when fishing in the coastal waters is banned, the major fishing takes place in these estuarine regions where shellfish dominates during monsoon period. Further, it can be concluded that increase in the nutrient can lead to harmful algal blooms during the monsoon which may be detrimental to marine life and humans. Reduction of nutrient loading by reducing the anthropogenic activities and to avoid extensive use of fertilizers for agricultural purpose is suggested.