Iranian Scientific Fisheries Journal

fa مقاله علمی – پژوهشی:‌ اثر زیست‌‌توده سیانوباکتری Anabaena sp. حاوی سم Microcystin-LR، رشد یافته در غلظت‌‌های مختلف فسفر بر زئوپلانکتون Daphnia magna Effect of biomass of Anabaena sp. containing the microcystin-LR, grown at different phosphorus concentrations on the zooplankton, Daphnia magna بيولوژي آبزيان بيولوژي آبزيان پژوهشي Research سیانوباکتری‌های تولیدکننده سم به‌عنوان یکی از عوامل تهدیدکننده زنجیره‌های غذایی آبزیان شناخته می‌شوند. هدف از پژوهش حاضر، بررسی اثر حاد و مزمن زیست‌توده سیانوباکتریAnabaena sp.  حاوی سم Microcystin-LR، پرورش‌یافته در غلظت‌های مختلف فسفر، بر زئوپلانکتونDaphnia magna  بود. بدین منظور، سیانوباکتریAnabaena sp.  به مدت ۱۰ روز در معرض غلظت‌های مختلف فسفر شامل غلظت 1/7 میلی‌‌گرم بر لیتر به‌‌عنوان تیمار شاهد و غلظت‌‌های 74/7، 38/8 و 66/9 میلی‌‌گرم بر لیتر فسفر در دمای 28 درجه سانتی‌‌گراد و نور فلورسنت با شدت 35 میکرومول فوتون بر ثانیه کشت داده شد. نتایج نشان داد که میزان تولید سم Microcystin-LR به‌ طور معنی‌داری تحت تأثیر غلظت فسفر قرار دارد (05/0>p) به‌طوری‌که بیشترین مقدار سم در تیمار 38/8 میلی‌‌گرم بر لیتر فسفر با 5/300 پیکوگرم بر میلی‌لیتر و کمترین مقدار آن در تیمار شاهد با 41/33 پیکوگرم بر میلی‌لیتر اندازه‌گیری شد. ارزیابی سمیت حاد زیست‌توده سیانوباکتری بر زئوپلانکتونD. magna  نشان داد که کمترین مقدار LC₅₀ ۹۶ ساعته (بیشترین سمیت) در تیمار 38/8 میلی گرم بر لیتر فسفر مشاهده شد که 12/271 میلی‌گرم وزن خشک زیست‌توده بر لیتر بود و با سایر تیمارها اختلاف معنی‌‌دار داشت (05/0>p). در آزمایش سمیت مزمن نیز بیشترین میزان مرگ‌ومیر در تیمار با 38/8 میلی‌‌گرم در لیتر فسفر با میزان زیست‌‌توده خشک 100 میلی‌‌گرم بر لیتر مشاهده شد که 60 درصد بود. هیچ مرگ‌ومیری در تیمار شاهد مشاهده نشد. بیشترین تعداد نوزادان در تیمار شاهد و کمترین تعداد نوزاد متولد شده نیز مربوط به تیمار 38/8 میلی‌‌گرم بر لیتر فسفر بود که به‌‌ترتیب 21 و 10 عدد شمارش شدند. اولین زمان تولیدمثل برای تیمار شاهد روز 8 و برای سایر تیمارها روز 9 ثبت گردید. نتایج حاصل بیانگر آن است که افزایش غلظت فسفر محیط می‌تواند از طریق افزایش تولید Microcystin-LR، سمیت زیست‌توده سیانوباکتریAnabaena sp.  را تشدید کرده و تهدیدی جدی برای زئوپلانکتون‌ها و پایداری اکوسیستم‌های آبی ایجاد کند. Introduction Eutrophication of freshwater ecosystems has led to a worldwide increase in the frequency of harmful cyanobacterial blooms (Paerl and Otten, 2013; Preece et al., 2017). Many cyanobacteria produce secondary metabolites known as cyanotoxins, among which microcystins are the most widespread and ecologically significant (Spoof and Catherine, 2017). Microcystin-LR (MC-LR) is recognized as the most toxic and prevalent microcystin variant in freshwater systems (Li et al., 2017). Species within the genus Anabaena are globally distributed and capable of producing microcystins under favorable environmental conditions (Welker and Döhren, 2006). Zooplankton—particularly cladocerans such as Daphnia magna—play a pivotal role in aquatic food webs and are widely regarded as sensitive indicators of cyanobacterial toxicity (Ferrao-Filho et al., 2008; Ger et al., 2016). Cyanotoxins are thought to act as chemical defenses against grazing, thereby constraining energy transfer from primary producers to higher trophic levels (Ferrao-Filho and Kozlowsky-Suzuki, 2011). In addition to their acute lethal effects, a growing body of evidence indicates that microcystins can induce a broad range of sublethal and chronic responses in zooplankton, including reduced feeding activity, impaired growth, delayed maturation, and diminished reproductive performance (Ferrao-Filho et al., 2000; Vilar et al., 2014). These chronic effects are ecologically significant because they may alter the population dynamics of primary consumers even at toxin concentrations below those causing immediate mortality (Ferrao-Filho and Kozlowsky-Suzuki, 2011). Moreover, prolonged exposure to cyanobacterial biomass can promote the accumulation of microcystins in zooplankton tissues, thereby increasing the potential for trophic transfer and biomagnification within aquatic food webs (Ferrao-Filho et al., 2014; Pham and Utsumi, 2018). Despite extensive research on cyanobacterial toxicity, relatively little is known about how phosphorus availability influences microcystin production and the resulting acute and chronic effects on zooplankton. Methodology Anabaena sp. was cultured under four phosphorus concentrations, including a control treatment corresponding to the basal BG-11 medium phosphorus (7.1 mg/L P) and three elevated phosphorus levels (7.74, 8.38 and 9.66 mg/L P). Cyanobacterial biomass was harvested, freeze-dried, and used for toxin quantification and toxicity experiments. Microcystin-LR concentrations were measured using an ELISA method following standard protocols (Fan et al., 2022). Acute toxicity tests were conducted using neonates (<24 h old) of D. magna exposed to different concentrations of freeze-dried cyanobacterial biomass (0–1000 mg dry weight L⁻¹) for 96 hours, and mortality was recorded periodically (Ferrao-Filho et al., 2014). Median lethal concentration (LC50) values were calculated using Probit analysis. Chronic toxicity tests were performed over a 15-day exposure period at lower biomass concentrations (12.5, 25, and 50 mg DW/L). Survival, time to first reproduction, and total offspring production were recorded according to established methods (Smutna et al., 2014; Herrera et al., 2015). At the end of the exposure period, microcystin-LR accumulation in D. magna tissues was quantified using ELISA. Results Phosphorus concentration significantly influenced microcystin-LR (MC-LR) production in Anabaena sp. cultured under different experimental treatments. ELISA analysis showed that MC-LR concentrations in freeze-dried cyanobacterial biomass ranged from 33.41 to 300.5 pg /mL. The highest MC-LR content was recorded in the treatment with 8.38 mg/L P, followed by 9.66 mg/L P, while the lowest toxin concentration was observed in the control treatment (7.1 mg/L P). Statistical analysis revealed significant differences among treatments (p < 0.05), indicating that phosphorus availability markedly affected toxin production. Acute toxicity assays demonstrated clear dose-dependent effects of cyanobacterial biomass on the survival of D. magna. Mortality increased with increasing biomass concentration in all treatments. Biomass derived from the 8.38 mg/L P treatment caused the highest mortality, reaching 100% at 1000 mg DW/L within 96 hours. At intermediate concentrations (500 and 250 mg DW/L), mortality rates of approximately 70% and 50%, respectively, were observed for the 8.38 mg/L P treatment. In contrast, the control treatment exhibited the lowest toxicity, with mortality rates of 40%, 20%, and 10% at biomass concentrations of 1000, 500, and 250 mg DW/L, respectively. The lowest 96-h LC50 value (271.12 mg DW/L) was observed for the 8.38 mg/L P treatment, whereas higher LC50 values were observed for the remaining treatments, indicating lower toxicity. Chronic toxicity experiments revealed significant lethal and sublethal effects of Anabaena sp. biomass on D. magna during the 15-day exposure period. Survival rates declined with increasing biomass concentration, particularly in treatments associated with higher phosphorus levels. The highest mortality rates were observed in the 8.38 and 9.66 mg/L P treatments at biomass concentrations of 50 and 100 mg DW/L. In contrast, no mortality was recorded in the control group without cyanobacterial biomass. Reproductive performance of D. magna was markedly affected by chronic exposure to cyanobacterial biomass. The total number of offspring produced over the experimental period decreased significantly with increasing biomass concentration in all treatments. The lowest cumulative number of neonates was recorded in the 8.38 mg/L P treatment, particularly at 100 mg DW/L, where reproduction was strongly suppressed. In contrast, the control group showed the highest reproductive output. Additionally, the time to first reproduction was delayed in all exposed groups compared to the control, with reproduction occurring one day later in treatments containing cyanobacterial biomass. Analysis of microcystin-LR accumulation in D. magna tissues indicated relatively low toxin concentrations at the end of the chronic exposure period. No detectable MC-LR was found in zooplankton exposed to 25 mg DW/L across all treatments. However, measurable toxin levels were detected at higher biomass concentrations, particularly in the 8.38 and 9.66 mg/L P treatments. The highest accumulation was observed at 100 mg DW/L, although concentrations remained low across all treatments. Discussion and conclusion The enhanced toxicity observed under phosphorus-enriched treatments can be attributed to increased microcystin-LR production and shifts in the biochemical composition of cyanobacterial biomass. Previous studies have shown that phosphorus can indirectly regulate microcystin production by promoting biomass accumulation and increasing cellular energy availability (Halstvedt et al., 2007; Wang et al., 2010). Phosphorus also plays a central role in ATP synthesis, providing the energy required for microcystin biosynthesis (Li et al., 2023). Chronic-exposure experiments revealed significant lethal and sublethal effects in D. magna, including reduced survival, delayed reproduction, and decreased offspring production. These responses are characteristic of chronic cyanobacterial toxicity and have been widely documented in cladocerans exposed to microcystins (Ferrao-Filho and Kozlowsky-Suzuki, 2011; Vilar et al., 2014). Although microcystin accumulation in D. magna tissues was relatively low overall, accumulation increased with prolonged exposure and under phosphorus-enriched conditions, consistent with previous reports of microcystin bioaccumulation and trophic transfer (Ferrao-Filho et al., 2014; Pham and Utsumi, 2018). Overall, these findings suggest that nutrient-driven cyanobacterial blooms may have long-term impacts on zooplankton populations through both direct toxicity and disruption of key life-history traits. In conclusion, cyanobacterial toxicity to D. magna appears to depend strongly on biomass dose and phosphorus-mediated toxin production, and chronic exposure may pose substantial ecological risks even at sublethal concentrations. Ultimately, these effects may compromise the population stability of primary consumers and reduce energy transfer in eutrophic freshwater ecosystems. Conflict of interest The authors declare no conflict of interest. Acknowledgement The authors gratefully acknowledge the Faculty of Natural Resources, Isfahan University of Technology, for providing laboratory facilities and technical support for this research. They also thank the faculty members and laboratory staff who assisted with the experimental work. Anabaena sp., Microcystin-LR, سمیت, Daphnia magna, فسفر Anabaena sp., Daphnia magna, Microcystin-LR, Phosphorus, Toxin 25 37 http://isfj.ir/browse.php?a_code=A-10-76-6&slc_lang=fa&sid=1 Fatemeh Rostami فاطمه رستمی rostami.1370@yahoo.com 100319475328460041856 100319475328460041856 No Department of Natural Resources, Isfahan University of Technology, Isfahan, Iran گروه شیلات، دانشکده منابع طبیعی، دانشگاه صنعتی اصفهان، اصفهان، ایران Omidvar Farhadian امیدوار فرهادیان omfarhad@iut.ac.ir 100319475328460041855 100319475328460041855 Yes Department of Natural Resources, Isfahan University of Technology, Isfahan, Iran گروه شیلات، دانشکده منابع طبیعی، دانشگاه صنعتی اصفهان، اصفهان، ایران Nasrollah Mahboobi Soofiani نصراله محبوبی صوفیانی soofiani@iut.ac.ir 100319475328460041857 100319475328460041857 No Department of Natural Resources, Isfahan University of Technology, Isfahan, Iran گروه شیلات، دانشکده منابع طبیعی، دانشگاه صنعتی اصفهان، اصفهان، ایران