Society of Environmental Toxicology and Chemistry (SETAC) Europe Conference Monday May 1st 2023
Introduction: context and choice of organism
Heart rate studies have been performed on crustaceans since their internal structure was seen to have an oscillating heart-like structure in the early days of microscopy. In 1894, J.W. Pickering (Pickering, 1894) used Daphniae [sic.] to explore the effects of a range of medications on simple cardiac structures, and specifically to mimic experiments in vertebrate embryos. By the third decade of the 20th century, Daphnia was established as a model of pharmaceutical studies, so much so, that Viehoever (1936) termed Daphnia “the biological reagent”. More detail on the overall history of biological studies into Daphnia may be found in Shaw et al. (2008) however suffice it to say that Daphnia and its relatives including Moina would form an indispensable tool in toxicological, pollution, and pharmaceutical research. So important was the role of Daphniids in research that as molecular techniques improved, the next natural step would be for the Daphnia whole genome to be mapped (Shaw et al., 2008). The need for simple, inexpensive methods for the determination of the role of effects-based pollution continues and not without further developments and innovations.
Although Daphnia is considered simple to culture, culturing Daphnia can be problematic as it requires a parallel algal culture for food and it can be sensitive to dissolved oxygen levels and pH of the medium. An organism similar to Daphnia, and to some extent easier to handle, is Moina macrocopa Strauss 1820(formerly Daphnia macrocopa) which tolerates poorer water conditions and which thrives on dried food such as ‘spirulina’ (powdered cyanobacteria: Arthrospira spp.) or on algae cultured within their own culture tank as with Daphnia.
Recent studies to measure heart rate in daphniids have included the following: Villegas-Navarro et al. (2003), Campbell et al. (2004), Chung et al. (2016), Lari et al. (2017), Fekete-Kertész et al. (2018). Zang et al. (2019) examined the ‘classic’ (after Pickering, 1894) caffeine effects on D. magna and found that caffeine and acetylcholine both decrease D. magna heart rate. A number of modern studies have examined cardiac flow rate (systolic and diastolic volumes) of Moina or Daphnia employ computerized video analysis (e.g. Santoso et al., 2020). The current study aimed to replicate similar experiments by analysing the heart rate of Moina macrocopa (n=30) following acute (48h) exposure to varying concentrations of diclofenac (2-[2-(2,6-dichloroanilino) phenyl]acetic acid; CAS ID: 15307-86-5) or DCF. DCF was listed in Decision 2015/495 on March 20, 2015. However, rather than detecting the presence of, and recording the levels of DCF in waters, it is incumbent upon researchers to determine specific effects on living organisms.
In addition to heart rate monitoring, other organs may be used for chronic effects, toleration and acclimation. For example, Consi et al. (1990) examined eye movements in Daphnia magna and found that different regions of the daphniid eye are specialized for different behaviours.
Methodology
A pilot study by JK (forthcoming) had shown sensitivity of Moina to paracetemol as a preliminary to this study. In addition, culturing methods were stabilised to allow this present study to proceed. Moina macrocopa Strauss 1820 were cultured in ‘spring’ water (BallygowanTM) and split into experimental and non-experimental cultures in the DCU Mesocosm. The cultures designated ‘experimental’ were doped with DCF to arrive at a concentration of .55 mg/L DCF. Following 48 hours, the survivors were investigated regarding cardiac function. Each organism was placed on a cavity glass microscope slide in a single drop of clean mineral water. The slide was mounted on the stage of an optical microscope (Olympus CH20BIMF200) with an attachable eyepiece camera (Optika, Italy) connected to an iPhone XR via cellular connection. The organism on the slide was brought into view and sharpened until the heart could be seen (a small transparent oval located dorsally of the hindgut closest to the anterior end of the organism).
If the organism was moving too much so as to make a recording impossible, enough water was removed with a pipette so as to immobilize the organism without causing death through acute dehydration. Care was also taken to avoid leaving the organisms under the light for prolonged periods before the recording so as to avoid any effect on heart rate through desiccation and unwanted mortality. Once brought into view, a 1 minute screen recording of the organism under the microscope was taken. All clips were recorded at 10x magnification. Following the collection of video clips, organisms were washed off the slide into a beaker and subjected to an acute heat treatment to ensure termination.
A series of baseline videos were prepared (by PM) in order to (re-)train the experimenter into counting heart-rates. The videos were slowed to 50% and 25% and segments of the videos were played during which a tally counter was used to number the beats and a stopwatch was used to time the segment. This was repeated until the error was low enough to consider the results ‘safe’, and usually required about 30 segments to be examined.
Once the experimenter (i.e., TMcC) was satisfied that the error was low enough (approximately 5%), the clips resulting from the treatment at .55 mg/l DCF and the control segments generated 30 video clips (although from n=3 individuals only) and 30 from the control specimens (n=4).
Different parts of the video were played to ensure a broad bandwidth (e.g., overcoming sudden movements by the specimens) and of varying lengths so to avoid subconscious bias.
Results
The results of the number of beats and length of clip examined (adjusting for the slowing of the playback) were recorded and a Student’s t-Test conducted.
The calculated value of t was found to be -2.076044 and the value of p was .04719. Therefore the result is significant at p < .05 (but was not at < .01) which demonstrated an average elevation in the heart rate of Moina macrocopa due to the concentration of .55 mg/L DCF. Repeated iterations throughout the series of clips produced the same result.
Conclusions
The chronic effects of the pharmaceutical were established by observing organ function and we report the effect of DCF on the rate and rhythm of the crustacean ‘heart’ as a function of overall life processes. DCF has an acute toxic effect on daphniids and further work is required to establish the differential in survival between Daphnia and Moina. Owing to the low numbers of surviving Moina after 48hrs (typically n=3 or 4 in each batch regardless of the concentrations of DCF), this preliminary study was hampered by the low number of survivors subjected to video analysis. a more fine-tuned experiment with a fresh culture and employing much larger numbers of individuals is recommended, typically n=30 at each concentration.
References
Campbell, A. K., Wann, K. T., Matthews, S. B. (2004) Lactose causes heart arrhythmia in the water flea Daphnia pulex. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology. 139: 225–234.
Chung, W.; Song, J.M.; Lee, J. (2016) The Evaluation of Titanium Dioxide Nanoparticle Effects on Cardiac and Swimming Performance of Daphnia magna. International Journal of Applied Environmental Science. 11, 1375–1385.
Consi, T.R.; Passani, M.B.; and Macagno, E.R. (1990) Eye movements in Daphnia magna. Regions of the eye are specialized for different behaviors. Journal of Comparative Physiology, Series A. 166(3): 411-420.
Fekete-Kertész, I.; Stirling, T.; Ullmann, O.; Farkas, É.; Kirchkeszner, C.; Feigl, V.; Molnár, M. (2018) How Does Experimental Design Modify the Result of Daphnia magna Heartbeat Rate Test?–Analyses of Factors Affecting the Sensitivity of the Test System. Periodica Polytechnica Chemical Engineering. 62: 257–264.
Lari, E.; Steinkey, D.; Pyle, G.G. (2017) A novel apparatus for evaluating contaminant effects on feeding activity and heart rate in Daphnia spp. Ecotoxicology and Environmental Safety 135: 381–386.
Pickering, J.W. (1894) On the action of certain substances on the hearts of Daphniae. Journal of Physiology 17(5): 356 – 359.
Santoso, F.; Krylov, V.V.; Castillo, A.L.; Saputra, F.; Chen, H.-M.; Lai, H.-T.; and Hsiao, C.-D. (2020) Cardiovascular Performance Measurement in Water Fleas by Utilizing High-Speed Videography and ImageJ Software and Its Application for Pesticide Toxicity Assessment. Animals, 10: 1587; doi:10.3390/ani10091587
Viehoever, A. (1936), Daphnia—the biological reagent. Journal of Pharmaceutical Science. 25(12): 1112- 1117.
Villegas-Navarro, A., Rosas, L. E., Reyes, J. L. (2003) The heart of Daphnia magna: effects of four cardioactive drugs. Comparative Biochemistry and Physiology Part C: Toxicology and Pharmacology. 136: 127–134.
Zang, C.; Laia, N.; Gruse, A.; Comfort, C.; Felder, M. (2019) Caffeine and acetylcholine decrease Daphnia magna heart rate. Journal of Undergraduate Biology Laboratory Investigations. 2, 2.
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