1 Dynamics and Toxicology of Microcystis and Anabaena and their Correlation

2 with Environmental Factors in Eutrophic Ponds

3 Sunzida Sultana,1 Saleha Khan,1* Zinia Rahman,2 Sadia Momota Hena,1 Md Sayem
4 Ahmed,1 Md Mahfuzul Haque,1 Yahia Mahmud3

5 1 Department of Fisheries Management, Bangladesh Agricultural University, Mymensingh,

6 2202, Bangladesh
7 2 Department of Genetics and Fish Breeding, Bangabandhu Sheikh Mujibur Rahman
8 Agricultural University, Gazipur 1706, Bangladesh
9 3 Bangladesh Fisheries Research Institute, Mymensingh 2201, Bangladesh

10 *Correspondence: salehakhan@bau.edu.bd (Saleha Khan)

11 Orcid ID and Email ID of All Authors

12 Sunzida Sultana https://orcid.org/0000-0002-6728-0989

13 E-mail: sunzida43812@bau.edu.bd

14 Saleha Khan https://orcid.org/0000-0002-1151-0052

15 E-mail: salehakhan@bau.edu.bd

16 Zinia Rahman

17 E-mail: zinia@bsmrau.edu.bd
18 Sadia Momota Hena https://orcid.org/0009-0001-3000-5262
19 E-mail: sadia.mfs@bau.edu.bd

20 Md Sayem Ahmed E-mail: ahmedsayem017@gmail.com

21 Md Mahfuzul Haque https://orcid.org/0000-0002-0452-6064

22 E-mail: mmhaque@bau.edu.bd
23 Yahia Mahmud

24 E-mail: yahiamahmud@yahoo.com

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27

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29 Abstract
30 As microalgal blooms are making serious threats in water bodies throughout Bangladesh, the
31 dynamics and toxicology of Microcystis and Anabaena in relation to environmental factors were
32 analyzed in two eutrophic ponds. The bloom of Cyanophyceae was dominated mainly by
33 Microcystis, Anabaena, Oscillatoria, Aphanothece, and Spirulina. Among them Microcystis was
34 the most dominantly occurring genus. Overabundance of Microcyctis may suppress the growth
35 of Anabaena and other microalgal species. In both ponds, seasonal succession of Microcyctis
36 was found dominant during the summer and the spring, whereas Anabaena was only
37 characterized in summer. Moderately higher temperature, nitrate-nitrogen, and phosphate-
38 phosphorus enrichment increased the biomass of both Microcystis and Anabaena. Vigorous
39 growth and abundance of Microcystis and Anabaena found to produce a dominant type of
40 cyanotoxin referred as microcystins in summer and spring. The high concentration of
41 microcystin in water could be a pernicious warning for aquatic organisms as well as human
42 health. The findings emphasized the necessity of regular investigation and observation of
43 microcystin-producing Microcystis and Anabaena in eutrophic ponds, which are also utilized for
44 household activities and drinking purposes, not only in Bangladesh but also in other developing
45 countries.

46 Keywords
47 Aquatic pollution, Seasonal dynamics, Toxicology; Microcystis, Anabaena, Microcystins.
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60 1. Introduction
61 Globally, aquatic pollution has become an alarming issue, including a worldwide decline and
62 collapse of fisheries [1]. Although microalgae are an essential component of aquatic ecosystem,
63 toxic and noxious algae play an alarming role in aquatic pollution [2]. Due to the increased
64 human population with technological advancement, industrialization, and urbanization, the
65 greater abundance of toxic and noxious algae has been initiated in water bodies during the last
66 decade [3]. Recently, cyanobacterial harmful algal blooms (CyanoHABs) are occurring more
67 frequently in eutrophic water bodies and have a variety of negative effects on the environment,
68 society, and the economy [4,5].

69 Cyanobacteria are prokaryotic photosynthetic organisms in eutrophic freshwater systems which

70 are also recognized as blue green algae and form surface scums [6-8]. Some species of
71 cyanobacteria are responsible for off-flavour in fish flesh because they have the ability to
72 synthesize chemical compounds such as geosmin and 2-methylisoborneol which are excreted
73 into the water and absorbed by fish, giving them an off-flavour [9-11]. The main bloom-forming
74 cyanobacterial taxa are Microcystis, Anabaena (now taxonomically recognized as
75 Dolichospermum), Aphanizomenon, Planktothrix, and Woronichinia [12]. Increasing density of
76 bloom-forming cyanobacteria in freshwater produce toxic metabolites and cyanotoxins,
77 including cyclic peptides (microcystins, nodularins) and alkaloids (anatoxins,
78 cylindrospermopsins, saxitoxins) that can be hepatotoxic, neurotoxic, genotoxic or cytotoxic
79 which have unthrifty effects on many aquatic animals, including zooplankton, zoobenthos, fish,
80 amphibians, aquatic birds, and aquatic plants [13-16]. Ingestion of cyanotoxin contaminated
81 water is highly hazardous and the main route of exposure to human and animal health [17,18].

82 Microcystis is one of the most pervasive and hazardous bloom-forming species among the
83 planktonic cyanobacteria. This alga is mainly capable of producing microcystin [19,20].
84 Microcystins are reported to be the most abundantly occurring cyanotoxins in freshwaters, and
85 Microcystis, Anabaena and Planktothrix species are typically responsible for the production of
86 microcystins [21]. However, data on the secretion of other cyanotoxins by this genus is scarce or
87 preliminary. Another bloom-forming harmful cyanobacterium is Anabaena, which is highly
88 responsible for harmful algal blooms and cyanotoxins production in waters worldwide [22-24].
89 Anatoxin-a and microcystin are the most commonly detected cyanobacterial neurotoxin from
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 90 Anabaena. It is also reported to cause ecological damage through high biomass and other related
91 detrimental effects [25].

92 During many decades, microcystin pose a serious risk to fisheries, livestock, pets, and wildlife
93 [26]. Reports of human illness (abdominal pain, diarrhea, and vomiting) from microcystins are
94 also well documented among those who use impaired water resources for drinking water
95 supplies, recreational activities, and fisheries [27,28]. In ponds of Bangladesh, the average
96 concentration of microcystins was found to be higher (> 10 μg/l) than the provisional WHO
97 guideline concentration (1 μg/l) [29]. Mortality of fishes, irritative bad odour from decayed algae
98 and off-odour from fish muscle even after cooking have been reported from rural fish ponds [29-
99 31]. Contact with high levels of microcystins has been found to contribute in eye, ear, stomach,
100 and skin diseases.

101 Environmental factors interact to regulate spatial and seasonal growth and succession of
102 microalgal populations [32]. Algae have definite temperature optima and tolerance ranges, which
103 interact with other parameters to cause seasonal succession. A set of ecological and
104 environmental factors including nutrients, temperature, light availability, and competition can
105 influence the growth of algae [33-36]. When climate and other environmental conditions are
106 favourable, Microcystis and Anabaena proliferate and form continuous blooms in ponds surface
107 water round the year. Mass abundances of cyanobacteria are mainly related to nutrient inputs
108 resulting from agricultural runoff, livestock, and human wastewater [37]. Generally, different
109 cyanobacterial species have different tolerance levels of temperature as well as nutrient
110 concentrations. These tolerance levels indicate the dominance of different species within
111 different seasons. Hence the seasonal changes in the Microcystis and Anabaena can be explained
112 in terms of variations in water temperature and competition for nutrients and light. Though the
113 different seasons of a year in Bangladesh are not so marked as those of temperate countries, yet,
114 Microcystis and Anabaena show significant interaction with environmental and hydrological
115 parameters for seasonal growth. With the progress of pond aquaculture, studies of the microalgae
116 occurring in pond have gained considerable importance. The qualitative and quantitative
117 variation of phytoplankton in different months is likely to have direct effect on fish growth. So,
118 their species composition and seasonal trends are of great value.

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119 Within few years of successive fish culture, most of the culture ponds became hypernutrified
120 which in extreme cases caused algal bloom by heavy algal population. Harmful bloom-forming
121 Microcystis and Anabaena are a serious problem for freshwater ecosystems all over the world. In
122 many countries, scientists have conducted substantial study on how to restrict the proliferation of
123 Microcystis and Anabaena in water bodies, particularly in eutrophic aquaculture ponds, in order
124 to enhance water quality, protect aquatic lives, maintain the quality of aquatic products and
125 safety of humans [38-40]. But, extensive research on seasonal dynamics and toxicology of
126 harmful Microcystis and Anabaena in eutrophic ponds of Bangladesh are incommensurate.
127 Hence the present study was aimed to investigate the quantitative and qualitative alterations of
128 cyanobacterial (blue-green algae) community, their seasonal succession and toxin production in
129 relation to environmental factors, with especial emphasis on Microcystis and Anabaena in
130 eutrophic ponds.

131 2. Materials and Methods

132 2.1. Study area

133 The study of dynamics and toxicology of Microcystis and Anabaena was conducted for a period
134 of 12 months from March, 2021 to February, 2022 in two rural fish ponds. The ponds were
135 located in Mymensingh (24⁰45’14″N and 90⁰24’11″E) region of Bangladesh. The area of the two
136 rectangular shape ponds are 50 decimal (Pond 1) and 15 decimal (Pond 2). In addition, the
137 average depth of pond 1 and 2 were 10ft and 6ft, respectively. Pond 1 was generally used for
138 domestic purposes while pond 2 was only used for fish culture. The ponds had no inlet or outlet
139 system and proper embankment. The ponds also found to receive domestic wastes and
140 decomposed organic nutrients from different sides round the year with maximum during the
141 rainy season.

142 2.2. Analysis of environmental parameters and chlorophyll-a

143 Environmental parameters (temperature, pH and dissolved oxygen (DO)) were measured using a
144 HI-9829 multi-parameter meter. The total alkalinity and free carbon dioxide (CO2) was measured
145 by titration method of APHA [78]. Water samples for determination of nutrients were collected
146 from sub-surface in clean black plastic bottles. The nutrients, nitrate-nitrogen (NO3-N) and
147 phosphate-phosphorus (PO4-P) were measured using spectrophotometric methods (DR/2010,
148 HACH, Loveland, CO, USA, Spectrophotometer) with Nitraver 5 and Phosver 3 powder pillows

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149 (high range chemical) for 10ml filtered water samples [32]. Chlorophyll-a was also estimated by
150 filtering water samples through 0.45-μm membrane filters 47 mm in diameter (Whatman
151 GF/CTM™, GE Healthcare Ltd.) and pigments were extracted using the same spectrophotometer
152 followed by the acetone extraction method [82].

153 2.3. Phytoplankton study

154 For the qualitative and quantitative phytoplankton study, a known volume of 10 liters of pond
155 water was passed through a conical plankton net (mesh size, 25 µm) and fixed in 10% buffered
156 formalin for identification. The enumeration of phytoplankton cells was carried out in a 1ml
157 Sedgewick-Rafter counting chamber (50 mm long, 20 mm wide and 1 mm deep) using an
158 Optima Biological microscope (G-206) and following the sedimentation method of Utermöhl
159 [77]. As massive bloom of cyanobacteria was observed in samples, Microcystis and Anabaena
160 colonies were counted designedly following the dilution technique. Counted results were
161 summarized as cells per liter. Phytoplankton was identified following relevant taxonomic
162 literature of phytoplankton [78-80]. Phytoplankton species were identified up to species level,
163 but quantified by genera.

164 2.4. Enzyme-linked immunosorbent assay

165 Phytoplankton samples were tested for analyzing microcystins (MCs) concentration using
166 Enzyme-linked Immunosorbent Assay. From each sample, 10 ml water was taken into a 12 ml
167 polyvinyl vial and 1/100 volume of 10 % sodium azide was added and frozen in a deep freezer.
168 The samples were freeze–thawed twice, and filtered via glass fiber filters (Whatman GF/C, 25
169 mm in diameter) and used for enzyme-linked immunosorbent assay (ELISA). The water samples
170 or microcystins LR (MC-LR) standard were diluted with an appropriate dilution of the anti-
171 MCLR MAb M8H5, and added to a 96-well microtiter plate coated with MC-LR-bovine serum
172 albumin conjugate. After washing, the bound MAb was detected with horseradish peroxidase-
173 labeled goat anti-mouse IgG (TAGO 4550) plus substrate (0.1 mg ml-1 3, 30-5, 50-tetramethyl
174 benzidine, 0.005 % H2O2 in 0.1 M acetate buffer pH 5), resulting in an absorption measurement
175 at 450 nm. The concentrations of MCs used when examining the ELISA data were an average of
176 two triplicate estimations expressed as microgram per liter (μg/l) by MC-LR [81].

177

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178 2.5 Statistical analysis

179 All the analysis were conducted using the SPSS (Statistical Packages for the Social Sciences)
180 and PAST V3 (Palaeontological Statistics) software at 0.05% and 0.01% significance level. The
181 canonical correspondence analysis (CCA) was performed to find out the controlling
182 environmental factors of Microcystis and Anabaena abundance. Cluster analysis was done to
183 elucidate the similarity of Microcystis and Anabaena abundance based on Bray-Curtis.

184 3. Results

185 3.1. Total microalgal population

186 The composition and diversity of microlagal species was studied in two eutrophic ponds where a
187 total of 33 genera of phytoplankton belonging to Cyanophyceae (7 genera), Euglenophyceae (3
188 genera), Cholorophyceae (15 genera), Bacillariophyceae (7 genera) and Rhodophyceae (1 genus)
189 were identified. Among the microalgal groups, Cyanophyceae was the most dominant and the
190 abundance of Chlorophyceae occupied second place throughout the study period. Their
191 dominance was due to their abundance in number, not in species.

192 3.2. Abundance of cyanobacteria

193 Cyanobacterial population varied among two ponds during the sampling period. The highest cell
194 density was recorded in May, whereas, the lowest was detected in August in both ponds (Figure
195 1). Cyanobacterial community comprised of 7 genera and 15 species of which Microcystis was
196 the most predominant genus. The percent composition of individual cyanobacterial genera in
197 pond 1 was as follows: Microcystis 75%, Anabaena 7%, Aphanothece 3%, Spirulina 13%,
198 Oscillatoria 2%, and each of Aphanizomenon and Gomphosphaeria less than 1%. In pond 2
199 phytoplankton compositions was Microcystis 92%, Anabaena 2%, Aphanothece 2%, Spirulina
200 2%, Oscillatoria 1%, Gomphosphaeria 1% and Aphanizomenon less than 1% (Figure 2). Among
201 the Microcystis samples, Microcystis aeruginosa, Microcystis flos-aquae, Microcystis natans,
202 and Microcystis botrys were mainly found. Anabaena arnoldii, Anabaena circinalis, and
203 Anabaena oscillarioides were mainly observed among Anabaena samples during the
204 investigation period.

205

206

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208 FIGURE 1: Monthly variation in abundance of cyanobacteria in the two ponds.

209

210 FIGURE 2: Percentage of individual cyanobacterial genus in the two ponds.

211 3.3. Environmental parameters and chlorophyll-a

212 The ranges and the mean values of the environmental parameters were found to vary in the
213 studied ponds (Table 1). The water temperature reached its highest value in July in pond 1
214 (32.9°C) and in May in pond 2 (34.5°C), whereas, the lowest value was recorded in January in
215 both pond 1 (19.43°C) and in pond 2 (22.56°C). The dissolved oxygen (DO) concentration

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216 varied temporally and ranged from 2.23-8.73 mg/l in pond 1 and 2.63-12.43 mg/l in pond 2, with
217 an average of 3.90 mg/l in pond 1 and 7.38 mg/l in pond 2. pH values were recorded in alkaline
218 range during the study period in both of the ponds. Total alkalinity was generally high compared
219 to optimum level. Total alkalinity in both ponds was found maximum in March and minimum in
220 August. NO3-N concentration was lowest in pond 1 (0.05 mg/l) and in pond 2 (0.005 mg/l) in
221 July, while highest levels were observed in pond 1 (0.36 mg/l) and in pond 2 (0.29 mg/l) in May.
222 Similarly, lowest concentration of PO4-P was detected in August in pond 1 (0.02 mg/l) and in
223 pond 2 (0.07 mg/l) while highest values were observed in May in pond 1 (0.45 mg/l) and pond 2
224 (0.53 mg/l). In pond 1, higher free CO2 was found in March and lower in November. Remarkable
225 variations of free CO2 were observed in pond 2 between 0 and 135.17 mg/l. The lowest value (0
226 mg/l) was observed in several months (April, May, December, and February) and the highest
227 value of free CO2 was recorded in March. During the study period, the values of chlorophyll-a
228 varied from 107.07-558.17 mg/l in pond 1 and 203.66 - 618.54 mg/l in pond 2, respectively. The
229 monthly values of chlorophyll-a were found to be highest as 558.17 mg/l in May in pond 1 and
230 as 618.54 mg/l in April in pond 2. The values of chlorophyll-a were found to be lowest as
231 107.07 mg/l in pond 1 and as 203.66 mg/l in pond 2 in August. The mean values of chlorophyll-
232 a among the two ponds were 334.29 mg/l and 362.86 mg/l.

233 TABLE 1 The mean values with standard deviation (SD) and ranges of different
234 physicochemical parameters in the two ponds.
Parameters Ponds Average SD Minimum Maximum
(Month) (Month)
Temperature (⁰C) Pond 1 27.43 4.93 19.43 (Jan) 32.9 (Jul)
Pond 2 28.44 3.86 22.56 (Jan) 34.5 (May)
DO (mg/l) Pond 1 3.90 2.08 2.23 (Sep) 8.73 (Jul)
Pond 2 7.38 3.57 2.63 (Jul, Aug) 12.43 (Apr)
pH Pond 1 8.04 0.37 7.53 (Sep) 8.66 (Jul)
Pond 2 8.70 0.67 7.43 (Aug) 9.9 (Feb)
Total Alkalinity (mg/l) Pond 1 141.38 10.99 128.66 (Aug) 165 (Mar)
Pond 2 129.91 38.02 74 (Aug) 198.66 (Mar)
NO3-N (mg/l) Pond 1 0.25 0.09 0.05 (Jul) 0.36 (May)
Pond 2 0.18 0.10 0.005 (Jul) 0.29 (May)
PO4-P (mg/l) Pond 1 0.18 0.16 0.02 (Aug) 0.45 (May)
Pond 2 0.20 0.12 0.07 (Aug) 0.53 (May)
Free CO2 (mg/l) Pond 1 35.04 31.22 6.99 (Nov) 103.87 (Mar)
Pond 2 24.99 43.55 0 (Apr, May, Dec, Feb) 135.17 (Mar)
Chlorophyll-a (mg/l) Pond 1 334.29 133.07 107.07 (Aug) 558.17 (May)

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 Pond 2 362.86 127.04 203.66 (Aug) 618.54 (Apr)
235

236 3.4. Influence of environmental parameters on abundance of Microcystis and Anabaena

237 Canonical Correspondence Analysis (CCA) was drawn between 8 environmental parameters,
238 Microcystis and Anabaena abundance in two ponds (Figures 3A and 3B). The length of arrow is
239 related to the importance of the explanatory variable in the ordination and arrow direction
240 apprises positive and negative relations. The abundance of Microcystis and Anabaena was
241 greatly related with environmental parameters and positively related with both axes. In pond 1,
242 the PO4-P expressed supreme influence and the total alkalinity, NO3-N, chlorophyll-a, pH and
243 free CO2 indicated higher impact while the temperature and dissolved oxygen (DO) presented
244 relatively lower effect on the abundance of Microcystis and Anabaena (Figure 3A). In pond 2,
245 the temperature, PO4-P, total alkalinity, dissolved oxygen (DO), chlorophyll-a and pH indicated
246 highest relationship while NO3-N and free CO2 exhibited moderate effect on the abundance of
247 Microcystis and Anabaena (Figure 3B).

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249 FIGURE 3: CCA biplot between dominant genera and environmental parameters in (A) pond 1
250 and (B) pond 2.

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251 In the present investigation, Microcystis was the most dominant species among the planktonic
252 cyanobacteria. The abundance of Microcystis started to appear from March and showed peak
253 bloom in May and June when the temperature was between 30.3 to 34.5°C in pond 1 and pond 2,
254 respectively (Figure 4A). Highest cell densities of Microcystis were observed to be related with
255 alkaline pH (Figure 4B).

256 Significant differences were observed in the abundance of Microcystis in relation to different
257 dissolved oxygen values among the ponds. The highest abundance was recorded in pond 1 when
258 dissolved oxygen concentration was lowest (Figure 4C). It was also found that the cell density of
259 Microcystis was highest in pond 2 where dissolved oxygen was highest. In moderate
260 concentration of total alkalinity in pond 1 (150 mg/l) and pond 2 (157 mg/l), Microcystis
261 exhibited highest density and became prevalent (Figure 4D).

262 Blooms of harmful algae were found to utilize a large amount of nutrients of the water bodies for
263 their excessive growth. The relationship between Microcystis abundance and nutrient
264 concentrations is shown in Figures 4E and 4F. During the heavy bloom of Microcystis, both
265 nitrate-nitrogen and phosphate-phosphorus were highest and after that, a decreasing trend of
266 nutrient concentration found with decreasing Microcystis cell density. Furthermore, it was
267 monitored that Microcystis played a dominant role to create bloom throughout the study period
268 and inhibited the growth of other microalgae due to the ability of toxin production.

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270 FIGURE 4: Influence of A) Temperature, B) pH, C) Dissolved oxygen, D) Total alkalinity, E)

271 NO3-N, and F) PO4-P on Microcystis abundance in the two ponds.

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272 Much low abundance of Anabaena was observed in comparison to Microcystis. This might be
273 due to inhibitory effect of Microcystis on other species. During the present study, Anabaena
274 showed the highest abundance and became sub-dominant when the water temperature was
275 highest in pond 1 and pond 2 (Figure 5A). Higher pH content (pH >7) of water was reported to
276 be the reason for a bloom of Anabaena (Figure 5B).

277 Dissolved oxygen ranged from 2.4 to 12.4 mg/l during the study period. The cell densities of
278 Anabaena were found high in low dissolved oxygen concentration in pond 1 while in pond 2, its
279 abundance was observed high in high dissolved oxygen concentration in May (Figure 5C). The
280 abundance of Anabaena was also found highest with the moderate concentration of total
281 alkalinity in pond 1 (150 mg/l) and pond 2 (157 mg/l) (Figure 5D).

282 In relation to the role of the nutrients on Anabaena abundance, it was observed that Anabaena
283 preferred higher nutrient concentration. The number of Anabaena was found to be increased with
284 the increasing level of NO3-N and PO4-P concentrations. High concentration of nitrate-nitrogen
285 as well as high concentration of phosphate-phosphorus was recorded during the maximum
286 growth of Anabaena in both ponds (Figure 5E and 5F).

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288 FIGURE 5: Influence of A) Temperature, B) pH, C) Dissolved oxygen, D) Total alkalinity, E)

289 NO3-N, and F) PO4-P on Anabaena abundance in the two ponds.
290

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291 3.6. Seasonal dynamics of Microcystis and Anabaena

292 The lowest abundance of cyanobacteria was observed in rainy season, while, the most
293 remarkable increases in cyanobacteria density took place in summer (Figure 6). During the study
294 period, Microcystis and Anabaena showed seasonal variation in both ponds. Microcystis was the
295 most abundant species throughout the monitoring period. In pond 1, the maximum abundance
296 was found in summer (963.33 x 105 cells/l) and in spring (676.67 x 105 cells/l) while the same
297 genera was most dominant only in summer (4561.67 x 105 cells/l) in pond 2. Contrariwise,
298 Anabaena showed its dominance along with Microcystis only in summer (217.67 x 105 cells/l) in
299 pond 1 (Figure 7). In pond 2, it was observed that dense scum of Microcystis suppressed the
300 maximum growth of Anabaena in different seasons.

301

302 FIGURE 6: Blue green algal blooms during summer season in the two ponds.

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304 FIGURE 7: Seasonal relative abundance of Microcystis and Anabaena in the two ponds.

305 In order to reveal the similarities among seasons, cluster analysis (CA) was carried out using the
306 Bray–Curtis similarity based on the total abundance of Microcystis and Anabaena (Figures 8a,
307 8b, 8c and 8d). At the similarity of 60%, three major clusters were found among seasons for
308 Anabaena in pond 1 (Figure 8a). The large cluster contained three seasons (summer, autumn, late
309 autumn), one cluster contained two seasons (rainy, spring) while another cluster (winter)
310 remained isolated. Two seasons (summer, late autumn) remained isolated at the similarity of
311 60%, while the largest cluster group contained four seasons of Microcystis in pond 1 (Figure 8b).
312 One season (summer) remained isolated, while one cluster contained two seasons (winter, late
313 autumn) and the largest cluster group contained three seasons (rainy, spring, autumn) of
314 Anabaena in pond 2 at the similarity of 30% (Figure 8c). At the similarity of 65%, three major
315 clusters were attained among seasons for Microcystis in pond 2 (Figure 8d). The large cluster

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316 contained three seasons (winter, rainy, late autumn), one cluster contained two seasons (summer,
317 spring) while another cluster (autumn) remained isolated.

318

319 FIGURE 8: Dendrogram showing clusters based on Bray–Curtis similarity matrix of 6 seasons
320 in (a) pond 1 for Anabaena, (b) pond 1 for Microcystis, (c) pond 2 for Anabaena, and (d) pond 2
321 for Microcystis.
322 3.7. Concentrations of microcystins (MCs)

323 The variation of MCs concentration with respect to cyanobacterial densities in different seasons
324 was investigated. It was found that when the bloom was ruled by Microcystis, MC was found

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325 constantly. MC occurrence may be related with the time when Microcystis subordinated the
326 buoyant blue green algae, and the maximum values were ordinarily combined with highest
327 Microcystis abundance.

328 The seasonal changes in MCs concentration in the water of the two ponds are shown in Figure 9.
329 In both ponds, microcystin was detected above 1 μg/l in all the seasons. Microcystin
330 concentrations were ranging from less than 1 to 22 μg/l in pond 1 and 3 to 65 μg/l in pond 2. The
331 highest concentrations of MCs were 22 μg/l at the peak bloom period of blue green algae
332 (1131.17 x 105 cells/l) in pond 1. In pond 2, MCs concentration was recorded at maximum level
333 (65 μg/l) when cyanobacterial cell densities were also at maximum (4997.83 x 105 cells/l) in
334 summer (Figures 9 and 10). Alongside in spring, MCs concentration was also highest 59 μg/l in
335 pond 2 when a thick surface scums of blue green algal bloom were observed during sampling.
336 Moreover, MCs concentration was found to occur less than 1 μg/l in rainy season in pond 1.

337

338 FIGURE 9: Seasonal dynamics of cyanobacteria in the two ponds.

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340 FIGURE 10: Seasonal concentrations of microcystins in the two ponds.

341 4. Discussion

342 For efficient management and successful culture practices, the aqua biologists always confront
343 the problems of productivity of water bodies. Microalgae plays a vital role in fisheries, but
344 excessive growth of harmful algae is always troublesome for fish production [41-43].
345 Eutrophication with heavy algal blooms, off-flavour in fish-flesh, high feed costs, reduced fish
346 growth comparing earlier years, gradual fall in consumer demand due to off-flavour development
347 in fish etc. are reported as the most important problems facing by the farmers [11,44]. Last few
348 decades, fish farmers are experiencing several unexpected problems like environmental
349 degradation with noxious blue green algal blooms and different fish diseases [45]. Microalgal
350 species diversity in eutrophic pond ecosystems is influenced by many biotic and abiotic

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351 processes. In the present study, a total number of 33 genera of microalgae were recorded
352 belonging to Cyanophyceae, Bacillariophyceae, Rhodophyceae, Euglenophyceae, and
353 Chlorophyceae. The dynamics of phytoplankton succession is a function of many of the
354 environmental processes that affect particular species diversity. Blue-green algae were observed
355 as the most dominant group among the phytoplankton and maintained its dominance throughout
356 the study period, which agrees with the findings of Rahman et al. [46]. Cyanophyceae was found
357 to be the most dominant group among the microalgae and maintained its dominance throughout
358 the study period, which agree with the findings of Begum et al. [47], Hossain et al. [48], and
359 Affan et al. [30]. The variation in number of species among the results of the different studies
360 could arise from the difference in the nutrient status, chemical qualities of water and density and
361 species composition of fish stocked.

362 The fish ponds, in which this study was carried out, was typically shallow, warmer and eutrophic
363 waterbodies that regularly produce dense blue green algal bloom from spring to summer in
364 Bangladesh. From March to June, 2021, a typical surface bloom of cyanobacteria started to be
365 observed in pond 1 whose intensity was very high and at that time the surface scum of
366 cyanobacteria covered most part of the pond. At the beginning of March 2021, cyanobacterial
367 bloom was found to be started in pond 2 and reached peak by the end of last week of May 2021.
368 During bloom, Microcystis was the most dominant genus which is consistent with Hossain et al.
369 [49] and Affan et al. [30] who also found Microcystis spp. dominant in their studies. Microcystis
370 represented 75 % in pond 1 and 92 % in pond 2 among the total Cyanophyceae. This result is
371 coherent with the findings of Oudra et al. [50] who recorded 95.00 % M. aeruginosa in a
372 cyanobacterial bloom in eutrophic Lalla Takerkousta reservoir in Morocco.

373 In the present study, Cyanophyceae comprised of 11 genera and the much higher abundance of
374 Microcystis was observed in comparison to other genera. This was might be due to inhibitory
375 effect of this genus on other genera. The inhibition of growth of Chlorella by Microcystis was
376 proved by the inverse correlation among them in all the ponds. Similar trend was recorded in
377 case of Euglena and Microcystis. Habib et al. [51] recorded that Microcystis and Anabaena had
378 inverse relation with most of the other microalgae. The inhibition of growth may be due to the
379 production of toxins of other microalgae which agrees with the findings of Lam and Silvester
380 [52]. Microcystis aeruginosa is one of the most cosmopolitan species among the planktonic
381 cyanobacteria. The optimum growth of algae was varied from species to species [53-55]. Several
21
382 environmental factors such as sunlight, temperature, pH, and nutrients influence the growth and
383 productivity of blue green algae [56]. The water temperature data from the two ponds revealed
384 the existence of a warm period from April to October (>29 °C). O’Neil et al. [57] confirmed that
385 in competitive environment, the growth rate of many cyanobacteria increases when temperatures
386 approach and exceed 20°C by stabilizing or decreasing the growth rates of freshwater eukaryotic
387 phytoplankton.

388 Microcystis aeruginosa was dominated by 95-98% of the total phytoplankton density during the
389 bloom. Rahman et al. [46] stated that the temperature for optimum growth of Microcystis is
390 between 29 and 30.5°C, so the recorded temperature during the present study was favourable for
391 the high concentration of the species. Murrell and Lores et al. [58] reported that M. aeruginosa
392 dominance often occurs when the temperature rises above 20°C. This finding is also similar to
393 Visser et al. [59] under culture condition. Another cause of higher abundance of M. aeruginosa
394 might be due to favourable hydrographical conditions.

395 Average DO were low during highest Microcystis and Anabaena abundance in both ponds with a
396 minimum of 2.23 mg/l recorded in September in pond 1 is supported by Wahab et al. [60]. pH
397 values were alkaline (>7) in two ponds throughout the study, which was parallel with elevated
398 cell abundance of Microcystis and Anabaena. Ye et al. [61] indicated that pH values are usually
399 higher during a bloom event due to cyanobacterial photosynthesis. Alkaline water may have
400 promoted the outbreak of Microcystis and Anabaena bloom. The alkalinity of water ranged from
401 128.66 to 165 mg/l in pond 1 and 74 to 198.66 mg/l in pond 2 during the bloom period agrees
402 fairly well with Boyd [62], who reported the alkalinity above 20 mg/l acceptable for pond water.
403 The higher values of nutrient in the two ponds might be related to the maximum abundance of
404 Microcystis and Anabaena. Highest nutrient (nitrate-nitrogen and phosphate-phosphorus)
405 concentrations were found at pond water during the highest abundance of Microcystis and
406 Anabaena. Similarly massive cyanobacterial cell density due to high nutrient concentrations was
407 found by Khan et al. [63], Jahan et al. [64], and Affan et al. [65] in eutrophic ponds of
408 Bangladesh.

409 Nitrate-nitrogen is one of the most important chemical parameters that influence the growth of
410 any alga [66,67]. Rahman et al. [46] suggested that high concentrations of NO3-N influenced the
411 growth as well as the toxin production by increasing M. aeruginosa propagation. The number of

22
412 M. aeruginosa was found to be increased with the increasing level of NO3-N and PO4-P
413 concentrations. Higher phosphorus content of surface water was reported to be the reason for a
414 bloom of the cyanobacteria, M. aeruginosa [65]. The inhibition of growth of other microalgae
415 may be due to the toxin produced by the Microcystis. Microcystis played a dominant role to
416 create bloom. Dominancy of Microcystis has also been reported by Habib et al. [51], Cunqi et al.
417 [68], and Jahan et al. [64].

418 Competition is a crucial factor promoting changes in the predominant algae in eutrophic water
419 bodies [69]. The algae that are coexisting with each other compete for light, nutrients, and other
420 resources. They may even produce compounds that prevent growth in order to hinder one
421 another's development [70,71]. Similarly, Microcystis and Anabaena showed the competition–
422 inhibition relationship in the present study. Microcystis highly absorbed nutrients and inhibited
423 the growth of Anabaena by altering cell size following cell density and the nutrient concentration
424 in the water bodies. Two peak periods of Microcystis bloom were found during the study period
425 in pond 2. One was in May-June (summer) and another was in March-April (spring). In
426 agreement with the present findings, Rahman and Jewel [72] found relatively high cell density of
427 cyanobacteria (dominated by M. aeruginosa) in fish culture ponds of Rajshahi City Corporation
428 in March (spring). Moreover, in different seasons, low abundance of Anabaena is associated with
429 high Microcystis abundance. Even though Anabaena was not a dominant genus, its high
430 abundance was detected with highest abundance of Microcystis, particularly in summer in the
431 two studied ponds when the temperature and nutrients concentration was also observed very
432 high. Similar findings were also reported by Affan et al. [31] and Li et al. [73]. The
433 consequences of high Anabaena and Microcystis abundance during summer was fish morality.
434 Thick algal blooms of cyanobacteria inhibit light penetration as well as they use most of the
435 nutrients from the water body for their growth. As a result the growth of other beneficial
436 planktons decreased markedly. Jewel et al. [45] reported a fish mortality concomitant to blue
437 green algal blooms and figured out the fish kill was possibly occurred by oxygen deficiency or
438 toxins secreted from a cyanobacteria or by the combination of both.

439 Cyanobacterial bloom often leads to algal die-off, shading of submerged vegetation, disruption
440 of food web dynamics and structure, and oxygen depletion that ultimately reduce fish
441 production. The link between fish mortality and the above mentioned genera are also supported
442 by Welker et al. [29], who detected that microcystin concentration in Bangladesh ponds ranged
23
443 between <0.1 and >1,000 μg/l, well above the WHO guideline value of 1 μg/l. So the results of
444 the present study more or less consistent to the finding of the above author. High concentrations
445 of MCs were found from the two studied pond water which are used for drinking, cooking and
446 other purposes. Local people who directly use the ponds water suffer from cholera, dysentery,
447 high fever, typhoid, hepatitis, eye inflammation and skin irritation. In china and South Africa,
448 hazardous concentrations of MCs in both natural and drinking water have been reported [74].
449 Pham et al. [75] and Dao et al. [76] points out that MCs concentration also exceed the WHO
450 guideline value of 1 μg/l in the Tri An and Dau Tieng reservoirs, similar to present study.
451 Microcystis was found as the main bloom-forming and toxin producing genus in the both ponds,
452 therefore it is strongly recommended to study further in more detail to elucidate the interactions
453 of Microcystis and Anabaena with environmental factors and mitigate the impact of toxin for
454 sustainable fisheries and human health in the future because the rural fish ponds of Bangladesh is
455 used by human for aquaculture and also for household activities.

456 5. Conclusions

457 The composition and succession of noxious blue-green algae in natural assemblage of eutrophic
458 ponds of tropical region was investigated. Efforts were made to study certain physicochemical
459 factors and their influence on the composition and seasonal abundance of these species. Various
460 environmental factors such as moderately high water temperature and high nutrient
461 concentrations of pond water found to create suitable condition for heavy growth of harmful
462 Microcystis and Anabaena. The seasonal cycles of Cyanophyceae in both eutrophic ponds
463 showed the classic pattern, being dominated by Microcystis sp. in the spring to summer and by
464 Anabaena sp. only in the summer. Microcystins concentrations were also found very high during
465 overabundance of Microcystis and Anabaena. The high ranges of microcystins in respective
466 waters could pose high risk on the pond ecosystems and human health. Therefore, further studies
467 on toxic effects of blue green algae on fish production, relationships between environmental
468 factors and cyanotoxins, and nutrient management system in fish ponds is required in different
469 regions of Bangladesh to keep the pond water danger-free from unexpected Microcystis and
470 Anabaena bloom and cyanotoxins.

471 Data Availability Statement

472 The data generated in the present study are available on request from the corresponding author.

24
473 Funding

474 This research was funded by University Grants Commission (UGC) of Bangladesh (Grant order
475 No. 37.01.0000.073.02.002.19 Date: 10.3.2020)

476 Conflicts of Interest

477 The authors declare that they have no known competing financial interests or personal
478 relationships that could have appeared to influence the work reported in this paper.

479 Author Contributions

480 Conceptualization, S.K.; methodology, S.S.; formal analysis, S.S.; investigation, S.K.; data
481 curation, S.S., S.M.H.; writing—original draft preparation, S.S.; writing—review and editing,
482 M.M.H., Y.M., Z.R., S.K.; supervision, S.K.; project administration, S.K.; funding acquisition,
483 S.K. All authors have read and agreed to the published version of the manuscript.

484 Acknowledgments

485 We are grateful to the pond owners who kindly allowed us to conduct our research in their
486 ponds.

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