Rock and water samples, control without D. geminata and with 12 bolons 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 all- 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 (Parodi et al. 2015), 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 collection points the different collection points, with 50% distilled water for a total volume of 14 L, leaving a 15 cm water column above the rocks. All artificial river from each location was 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 71009 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. 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 report 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-minute 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 minutes, 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 was performed using a total of 1 g of extract. Samples were diluted with Folin-Ciocalteu reagent following the protocol described by Lowry et al. (1951), and the polyphenol absorbance was measured at 517 nm. A 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). 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 hours 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 weeks. 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 describe previous on laboratory maintained. Then, neonates of D. magna organisms used in the test can be collected by filtration through a sieve (opening size of 560-µm for D. magna) or separated manually within 24 hours of birth. Then, 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 hours, and the mobile forms for each condition was recounted for an indication of viability after this time incubation. The control needed to show viability more significant than 80% to be considered correct for studying the viability of the different conditions, nine independent experiment was made for described the viability of D.magna on different experiment condition.
D. magna motility
The motility experiment is made using fresh D. magna, take for the cultured and deposit 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 condition, and we used 100 specimens in the total study of motility. We exposed in acute manner, at meditated manner 30 min of incubation or a chronic manner after 72 hrs of incubation, to the different condition. 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 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 has 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, has positive control.
Unless otherwise indicated, the results, including image analysis, are presented as the means ± SEM. Statistical comparisons were performed using Student’s t-test or ANOVA with the software graphpad prism 4. A probability level (p) of less than 0.05 was considered statistically significant.