Polyphenols extracts from Didymosphenia geminata (Lyngbye) Schmidt altered the motility and viability of Daphnia magna

The invasive diatom Didymosphenia geminata (Lyngbye) Schmidt, D. geminata has invaded the austral zone of Chile, causing significant ecological, scientific and societal concerns. We aimed to evaluate the viability and motility of Daphnia magna (D. magna), as a biosensor for effects of D. geminata. Toxicity assays were performed in dilutions of river water alone (V/V dilution) and in river water contaminated with D. geminata (V/V dilution) or polyphenols extracted from D. geminata under controlled conditions and different time (acute 30 min and 7 h). Our results indicated that D. magna was sensitive to increasing concentrations of D. geminata extracts. We observed a 50% (IC50) viability reduction after 24 h of exposure to a 0.023 V/V dilution and the same value when using polyphenols from D. geminata; additionally, this treatment further reduced the motility capacity by 50% after 72 h. The D. magna organisms were acutely responsive, showing a 50% reduction in frequency at 15 min. We conclude that D. magna is sensitive to polyphenols produced by D. geminata in rivers, suggesting potential chronic toxic consequences on several aquatic species following exposure to these diatom substances.


Introduction
Biological invasions of non-native species are a significant threat to biodiversity (Firn et al. 2015;Gurevitch and Padilla 2004) and socio-ecological systems . In Austral native freshwater ecosystems, the recent proliferation of Didymosphenia geminata (Lyngbye) Schmidt (D. geminata) has become a significant concern because of the effect on the ecosystem services they provide (Reid and Torres 2014;Strayer 2010;Taylor and Bothwell 2014). D. geminata blooms beat oligotrophic aquatic systems for several hundred kilometres (Montecino et al. 2014;Pinto Torres et al. 2016). It altered the physicochemical conditions of the river and the benthic fauna distribution, in particular macroinvertebrate communities (Brand and Grech 2020). It produced negative ecological and economic consequences (Alpert et al. 2000;Beville et al. 2012;Taylor and Bothwell 2014). Considerable attention has been paid to this D. geminata problem, as it has invaded more than 187 rivers in many other countries (Blanco and Ector 2009;Gretz et al. 2007). This benthic diatom has been declaring an invasive species in Southern Hemisphere countries such as New Zealand (Kilroy and Unwin 2011), Argentina  and Chile (Reid and Torres 2014) since 2010 (Segura 2011). Under favourable environmental conditions, it is capable of producing blooms with large amounts of extracellular stalks in a short period and colonises far from its geographical range (Bishop and Spaulding 2017;Cullis et al. 2012). In the Southern Hemisphere, D. geminata has shown more aggressive performance, with a considerable impact due to the extensive formation of biomass (Kilroy et al. 2009), which is likely caused by the favourable climate and physicochemical water conditions (Kunza et al. 2018), allowing it to bloom over the river rocks or be present but not invading in the planktonic phase (Montecino et al. 2016).
The most commonly described D. geminata impacts are physicochemical changes in the watercourses in oligotrophic locations, including substantial increases in algal biomass, the retention of fine sediment, and benthic hydrodynamic alterations, which consequently affect biogeochemical states and processes such as redox conditions, pH and nutrient cycling in the benthic layer (Reid et al. 2012). Other impacts are described in the periphyton biomass and benthic communities, showing higher tolerant invertebrate groups densities such as Oligochaeta Chironomidae, Cladocera and Nematoda, Orthocladiinae and also non-insect taxa (Brand and Grech 2020;Kilroy et al. 2009).
The toxic impacts of this microalgae start with the alteration of microenvironments and microalgal communities according to the seasonal variations of D. geminata (Chester and Norris 2006;Figueroa et al. 2018) and the possibility of these microalgae spreading to new bodies of water (Montecino et al. 2014;Reid et al. 2012) through different vectors (Leone et al. 2014). More observational and experimental work suggests that D. geminata is causing direct and indirect effects through the whole food web Parodi et al. 2015), during periphyton blooms (Chester and Norris 2006;Suren et al. 2003) with D. geminata (Kilroy et al. 2009) as well as after its removal (Larned and Kilroy 2014).
D. geminata toxicological potential to affect other organisms remains unknown. Moreover, this pennate microalgae is rich in antioxidants such as polyphenols and pigments including diadinoxanthin (Lohr and Wilhelm 1999), which was also reported as D. geminata polyphenol toxicity on two salmonid species cell lines (Olivares-Ferretti et al. 2019) and Salmo salar spermatozoa activation times , in this previous work the polyphenol are suggested the mechanism for generated toxic effect. In a large-scale ecological change in freshwater ecosystems, perturbations to macroscopic organisms are often documented (Ricciardi and MacIsaac 2000), suggesting the possibility that D. geminata polyphenol toxicity affected the behaviour of benthic bioindicator organisms like Daphnia magna (D. magna) and used this model to explore the toxicity (Kim et al. 2003), as an excellent tool for assessment of this problem (Jellyman et al. 2011). D. magna, due to its short life cycle, morphological characteristics and reproductive capacity, is a suitable organism for the study of toxins in aquatic environments (Baun et al. 2008;Gerhardt 2007). Our study aimed to elucidate the toxicity levels and effects of D. geminata polyphenols on D. magna, like new step in the trophic chain in aquatic ecosystem, measured viability and motility using the frequency of movement as a parameter for evidence of sublethal effects.

Sample's collection
Rock and water samples, control without D. geminata and with 12 round flat stones and with D. geminata were collected from each of three rivers from central and south Chile during the autumn and winter of 2019 as follows: Bio-Bio River, W318358, N5718384, W304806a and Point Lamin, W307366, N5713357N5713357; Espolón River at Futaleufú Point, W2667125, N52413723; Futaleufú River at Yelcho Point, W269107, N5213938. The samples were provided by the Chilean National Fishery Services (SERNAPESCA). After collection, the samples were immediately transported to the LaBCeMA laboratory, Universidad Mayor Temuco. A mixture of water and rocks with D. geminata from each river was stored in coolers and maintained at 4°C until arrival at the laboratory.

D. geminata laboratory maintenance
The samples were distributed in an implemented ''artificial river'' recirculation system  using control water, without D. geminata samples and water contaminated to generate a closed system of water contaminated with D. geminata for six months of collection according to the SERNAPESCA biosafety protocol for laboratory assays (Authorisation No. 3500). We followed the methodology standardised by Parodi et al. (2015). Briefly, the artificial river system was prepared by mixing 50% original river water from each of the different collection points, with 50% distilled water for a total volume of 14 L, leaving a 15 cm water column above the rocks. Artificial rivers from each location were maintained under a controlled temperature of 12°C using an expanded polystyrene insulating cover and a refrigerating gel system. The flow rates (1200 L/h) were controlled using a Plaset-Italy Model 71,009 recirculation engine, which maintained a steady flow and aeration. Macroscopic and microscopic changes in the artificial river systems were recorded daily for six months each year, as compared whit initial time, like a previous report of our group. The viability of the D. geminata population was observed with neutral red staining, and enough material was cultured to generate a 1 g polyphenol extract from 10 ml of wet D. geminata for use in the subsequent procedures and experiments.

Polyphenol extraction and liquid chromatography (HPLC) peak detection
In previous reports, we used the follow protocol (Olivares-Ferretti et al. 2019). A total composed wet D. geminata sample (10 ml) was obtained from two to five rocks collected (n = 12) and maintained in a single artificial river; no cultures of the sample were used, only fresh material was used, and it was exposed to liquid nitrogen. Samples from each point (10 ml) were macerated, and the cell frustules were ruptured by sonication (Misonix XL2000 Series) in 30-min pulses with one-minute intervals until the biomass (even complex samples) was wholly homogenised. A total of 10 ml distilled water was then added to the macerated samples, and the samples were collected in 15-ml tubes. The tubes were then incubated at 30°C under agitation for 20 min, filtered through double gauze and a Whatman No. 2 (125 mm) filter, and collected in a 20-ml glass flask (following Jofre-Fernandez et al. 2013). Finally, polyphenol detection (Total antioxidant measurement) was performed using a total of 1 g of extract. Samples were diluted with Folin-Ciocalteu reagent following the protocol described by Lowry (Lowry et al. 1951), for measured protein, but used for polyphenol detection, and the polyphenol absorbance was measured at 517 nm. Finally, total of 12 samples per year were frozen to avoid degradation until analysis by the HPLC service at the Universidad Austral de Chile to identify their profiles (Lohr and Wilhelm 1999) for detection of polyphenol in the sample. The presence of organic compounds was detected in the yellow fraction of the samples. A total of 5 different extracts from different collection points were used for the retention time measurements. A description of the antioxidant profiles of the samples and the absorbance at 440 nm with the AC18 column were determined (Macherey-Nagel, Duren, Germany).

Daphnia magna laboratory maintenance
D. magna specimens were used according to the NCh 2083 standard: With constant temperature 16°C, with a light/dark cycle (12 h/12 h). The culture water for D. magna was prepared with 25 ml saline solutions (calcium chloride: 11.76 gr/L; magnesium sulphate: 4.93 gr/L; sodium bicarbonate: 2.59 gr/L; potassium chloride: 0.23 gr/L) and filled up to 1000 ml with distilled filtered water. Finally, the water was aerated for 24 h before use. The growing culture density was from 10 to 15 D. magna organisms per 200 ml of water. D. magna were fed with 5 ml of the microalgae Chlorella vulgaris and Selenastrum capricornutum (Raphidocelis subcapitata) 30 ml/L per 500 ml of culture (25-35 D. magna) every 2 days. The approximate life cycle of D. magna is 3 to 4 months. The time to mature from juveniles to adults is approximately 15 days.

D. magna viability
D. magna specimens from at least the third generation were obtained by acyclic parthenogenesis under specific growing conditions described previously. Then, neonates of D. magna organisms used in the test were collected by filtration through a sieve (opening size of 560-lm for D. magna) or separated manually within 24 h of birth. Next, ten specimens for conditioning were split into an individual glass with 50 ml of artificial river water with oxygen. The specimens were exposed to the condition for 24 to 72 h, and the mobile forms for each condition were recounted for an indication of viability after this incubation time. The control needed to show viability more significant than 80% to be considered correct for studying the viability of the different conditions. Nine independent experiments were made to describe the viability of D.magna in different experimental conditions.

D. magna motility
The motility experiment was done using fresh D. magna, taking the culture and depositing it in a volume of 1 ml of artificial river water; 5 adults of D. magna were used in 9 different experiments for all the experimental conditions, and we used 100 specimens in the total study of motility. We performed acute exposures, in mediated style with 30 min of incubation or chronic style with 72 h of incubation, to the different conditions. We measured the absorbance at 328 nm on a Peak C-7100 Series instrument spectrophotometer. The absorbance peaks were continuously recorded for two minutes, as an indirect indication of the passage of the D. magna versus the light emission. The number of peaks per unit of time was counted, and the frequency of events was calculated as an indication of the motility event. Under these conditions, five adults were exposed to increasing concentrations of V/V of river water contaminated with D. geminata, and we used a river water without D.geminata as control. Increasing concentrations of polyphenols extracted from D. geminata between 5 and 500 ppm were used, and gallic acid (1 to 1000 ppm) was added to an artificial river water solution, as positive control.

Data analysis
Unless otherwise indicated, the results, including image analysis, are presented as the means ± SEM. The curve is made with log (inhibitor) vs. normalized response, and we obtained the IC 50 from this equation. Statistical comparisons were performed using Student's t-test or ANOVA, and Bonferroni post hoc test was applicated with the software GraphPad prism 4. A probability level (p) of less than 0.05 was considered statistically significant.

D. geminata-contaminated water effects on D. magna viability
The newborn and adult forms of D. magna were exposed to dilution by water contaminated with D. geminata and polyphenol extract from D. geminata and gallic acid. Figure 1a shows a curve of the concentration dilution V/V of the river water (Negative Control) or contaminated with D. geminata. The figure shows a reduction of over 50% in the percentage of life when the concentration of the river water contaminated with D. geminata is increased, with an IC 50 of 0.023 V/V. We explored the effect of polyphenols obtained from D. geminata and observed a decrease in viability (Fig. 1b) when the polyphenols were increased, with an IC 50 of 52 ppm. We compared the effect with a polyphenol standard, i.e. gallic acid (Positive control). Figure 1b shows the inhibition of viability when the concentration is increased, with an IC 50 of 4.5 ppm. Finally, we exposed D. magna to the IC 50 values in a chronic manner from the previous experiment, 72 h of incubation (Fig. 1c), and we observed a reduction in viability over 50% when D. magna was exposed to river water with D. geminata, a polyphenol from D. geminata or gallic acid.

Effect of D. geminata-contaminated water on D. magna motility
We explored the impact of the polyphenol obtained from D. geminata on the motility of D. magna. In  Fig. 2b when D. magna are exposed to increased concentrations of the D. geminata polyphenol extract; reduction form 0.1 Hrs to 0.05 Hz. The figure shows a reduction in the frequency of the peak when the concentration of polyphenol increases above 50 ppm, with an IC 50 of 52 ppm. We used this concentration to explore the time effect. We exposed the samples to 52 ppm of polyphenol extract from D. geminata at different times. In Fig. 2c, we show example traces of the absorbance peak, and the relation between absorbance and time. Figure 2d shows the quantification of motility at different times. We observed that 52 ppm did not induce a change in the frequency of events when used acutely; however, when D. magna were incubated longer than 5 min, the frequency of activity was reduced.

Effect of D. geminata-contaminated water on D. magna motility after acute incubation
We used the previous IC 50 value from water contaminated with D. geminata (0.023 V/V) polyphenol extract (52 ppm) and gallic acid (4.5 ppm) to observe the change in the frequency of D. magna after incubation for 30 min. Figure 3a shows examples of trace absorption changes in the relation between absorbance and time. Figure 3b shows motility quantification, and we observed a reduction of over 40% in the frequency when the D. magna samples were exposed to the different compounds. River water contaminated with D. geminata reduced the frequency by over 40%, and the effects of the polyphenol extract from D. geminata and gallic acid were similar.

Effect of D. geminata-contaminated water on D. magna motility after chronic incubation
We experimented again using the previous IC 50 value from water contaminated with D. geminata (0.023 V/  Figure 4a shows examples of traces of the absorption change. The relation between absorbance and time also shows an event over 10 when the control condition is observed and reduction to 5 events when polyphenols were used. Figure 4b shows the quantification of motility, and we can observe over 50% of reduction in the frequency when D. magna samples were exposed to the different compounds. River water contaminated with D. geminata decreased almost 50% of the frequency, and the effects of the polyphenol extract from D. geminata and gallic acid were similar.

Discussion
Our results presented in this work continue the results in previous studies about D. geminata toxicity and suggest the polyphenols present in the river water contaminated with the microalgae induce cell alteration, cellular death and now reduction in D. magna viability. The data present suggested a complex mechanism of the toxicity on the river, when are contaminated with D.geminata, and more future experiments are required for explain the pathway of this process. Since the introduction of D. geminata in local freshwater ecosystems, it has been a cause of concern to potential consequences on stream communities (Ladrera et al. 2018). In attempts to understand the D. geminata toxicity in a small organism, we have studied its effect on a standardised model. In our previous group research , we proposed that the toxic effects are secondary to the presence of polyphenols in river water contaminated with D. geminata. Our results suggest that water contaminated with D. geminata can be mediated to reduce effects on D. magna viability and motility by the presence of polyphenols, and in recent work, we control, Negative control (River water) water contaminated (Didymo) to polyphenol extract (poly didymo) or gallic acid (Galic). Each bar represents measurements from at least 5 independent experiments (mean ± SEM). The asterisk indicates p \ 0.05 (ANOVA) suggested that toxic effects at the cellular level are secondary to the present of polyphenols present in the river contaminated with D. geminata (Olivares-Ferretti et al. 2019). The experiment design in the present work utilised the D. magna standardised model to evaluate the effects of artificial river water contaminated with D. geminata, which was previously reported . The water samples included the presence of a biological compound that has been observed in D. geminata cells, where the pigment was measured at 440 nm identified by HPLC (Olivares-Ferretti et al. 2019) and is present in our samples. Previous studies have reported the D. geminata effects on aquatic organisms and spermatozoa (Brand and Grech 2020;Larned and Kilroy 2014;Olivares et al. 2015). Viability observations in river water with increasing V/V concentrations resulted in typical D. magna values; however, the level of D.
magna mortality increased when the river water was contaminated with D. geminata (Fig. 1a). We observed the lethal doses of river water contaminated with D. geminata and polyphenols from D. geminata and used a standard, gallic acid (Fig. 1b). It has been reported that due to the presence of epiphytic cyanobacteria in blooms, which are microcystin promoters (Whitton et al. 2009), higher microcystin dosage on D. magna caused chronic toxicity (Chen et al. 2005). Our results suggest that river contamination with D. geminata may have a chronic effect on aquatic organisms (Fig. 1c).
However, we followed the idea of subtoxic effects, and in particular, we explored a mechanism to explain the deadly impact. We evaluated D. magna motility using its endogenous fluorescence particularity by a UV light protocol, recording the absorbance of the solution, not explored the ionic change, has previous control, Negative control (River water) water contaminated (Didymo) to polyphenol extract (poly didymo) or gallic acid (Galic). Each bar represents measurements from at least 5 independent experiments (mean ± SEM). The asterisk indicates p \ 0.05 (ANOVA) report on the model, only follow the kinetic and suggest a mechanism secondary to concentration of polyphenols on the solution (Teplova et al. 2010). The traces obtained from the spectrophotometer (Fig. 2a  and c) show every change registered, considering the peaks as a movement of D. magna. We observed a reduced D. magna motility when the concentration of the polyphenol extracts from D. geminata was increased in an acute manner (Fig. 2b). An increasing basic metabolism in D. magna has been reported to counteract a prolonged exposure of cytotoxins in freshwater ecosystems, and help to suggested a complex model of receptors on the model, we not explored if the polyphenols increased signalling has in previous report, but we indicated relation of the motility and the doses of polyphenols (Grzesiuk et al. 2018). Our results showed reduced motility when D. magna was incubated with polyphenol DL 50 for a longer period of time (Fig. 2d). We suggest that D. geminata and the polyphenol produced by D. geminata alter motility in D. magna, and this can led to reduced viability in longer exposition, like in natural river.
The toxicity biotests were carried out by checking organism parameters (Walker et al. 2012). We recorded whether this effect occurred when river water contaminated with D. geminata. A standard sample of polyphenols and acid gallic was used (Maheshwari et al. 2017) at acute and chronic levels. Figure 3 shows a reduction on the D. magna motility in all the conditions; likewise, gallic acid had a stronger effect on motility, supporting the idea that the polyphenols had an effect on this parameter. A hypolocomotion response to water-born toxicants in D. magna has been described (Huang et al. 2017). Our data suggest an acute impact on motility mediated by the polyphenol present in the river water contaminated with D. geminata.
Anthropogenic influence generates biodiversity loss by increasing toxins in natural freshwater (Tickner et al. 2020), allowing us to consider the possibility of toxic components from the D. geminata invasion. Our data show reduced D. magna motility without a more substantial impact, indicating that the chronic effects at sublethal doses were the same as those observed in the acute experiment, which suggests that the effects on viability are more complex and future experiment help to explain a pathway of this effect observed (Fig. 4). D. geminata changes in macroinvertebrate composition blooms have been reported to show a decreased density in determinate trophic groups (Larned and Kilroy 2014;Whitton et al. 2009). However, the D. magna enzymatic metabolism increase to chronic toxicity adaptation mechanism has also been described (Chen et al. 2005). These effects are of great interest when considering whether the same effects are noted in the cell lines of native freshwater species, other species or biological models when representing a bioindicator for water quality (Venugopal 2002) and when evaluating whether D. geminata contamination can induce changes in river biota (Cifuentes et al. 2012;Montoya et al. 2012). Furthermore, there are few records of the biota or species being affected by microalgae in Chilean rivers or other places (Ladrera et al. 2018), representing a lack of research on the effects of D. geminata in cell models or using native river species. For example, a recent study indicated a change in the microalgae composition when D. geminata was present in a river (Zamorano et al. 2019).

Conclusion
Our study aimed to assess the harmful effects of D. geminata at the macroinvertebrate level, as well as the other effects of this diatom as observed in a previous report from our group. Our data propose that at sublethal concentrations, the presence of D. geminata polyphenols affects the viability of D. magna, due to inducing a reduction of the motility clouding when long-term exposure occurs. We also suggest a mechanism to explain toxicity among macroinvertebrates present in rivers contaminated with D. geminata. Our results suggest toxic and complicated implications of D. geminata contamination; specifically, the effect of polyphenols in D. geminata had a direct impact on macroinvertebrates in rivers and finally affected all the biota of the river.

Declarations
Conflict of interest The authors declare that they have no conflicts of interest.
Ethical approval This article does not include any studies with animals or human participants performed by any of the authors.
Human and animal rights No animals or humans were used in the present work.