Interactions between phenanthrene exposure and historical chemical stress: Implications for fitness and ecological resilience of the sentinel species Daphnia magna
Data files
May 21, 2024 version files 77.49 KB
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Gigl_et_al_chronic_toxicity.xlsx
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Gigl_etal_acute_toxicity.xlsx
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README.md
Abstract
Polycyclic aromatic hydrocarbons (PAHs) arise from incomplete combustion of oil, coal, and gasoline, with lipophilic properties facilitating their widespread distribution and persistence. Due to their biochemical attributes, PAHs can accumulate in animal tissues, potentially causing mutagenic and carcinogenic effects. Since the industrial revolution, PAH concentrations in the environment have risen, with some areas showing levels from 0.159 to 33,090 µg/kg sediment. Despite acute toxicity studies showing adverse effects on freshwater organisms, the long-term impacts and synergistic interactions with other pollutants remain largely unexplored.
This study investigates the impact of phenanthrene (PHE), a prominent PAH found in aquatic environments, on Daphnia magna, a species of significant ecological importance in freshwater ecosystems globally, being both a sentinel species for chemical pollution and a keystone organism in freshwater aquatic ecosystems. Leveraging the dormancy of D. magna, which spans decades or even centuries, we exposed strains with diverse histories of chemical contaminant exposure to environmentally relevant concentrations of PHE. Initially, acute exposure experiments were conducted in accordance with OECD guidelines across 16 Daphnia strains, revealing substantial variation in acute toxic responses, with strains naïve to chemical pollutants showing the lowest toxicity. Utilizing the median effect concentration EC10 derived from acute exposures, we assessed the impacts of chronic PHE exposure on the life history traits and ecological endpoints of the 16 strains. To elucidate how historical exposure to other environmental stressors may modulate the toxicity of PHE, temporal populations of D. magna resurrected from a lake with a well-documented century-spanning history of environmental impact were utilized. Our findings demonstrate that PHE exposure induces developmental failure, delays sexual maturation, and reduces adult size in Daphnia. Populations of Daphnia historically exposed to chemical stress exhibited significantly greater fitness impacts compared to naïve populations. This study provides crucial insights into the augmented effects of PAHs interacting with other environmental stressors.
README
- Gigl_etal_chronic toxicity. Fitness-linked life history traits collected following exposure to phenanthrene (PHE). GenotypeID; replicate; lake phase; treatment; age at maturity; size at maturity; day of release and size of first and second brood; overall fecundity expresses as number of juveniles in first and second brood combined; and mortality are shown. If mortality occurred before sexual maturity, life history traits linked to later life stages were not recorded (‘null’). If a mortality event does not occur and the life history traits are not recorded (‘null’), the individual is classified as a male.
- Gigl_etal_acute toxicity: GenotypeID; replicate; lake phase; treatment (Phenanthrene concentration); percentage of immobilized Daphnia after 24h and 48h are shown.
n/a: not available
Methods
Study system: The Daphnia genotypes used in this study were previously resurrected from the sedimentary archive of Lake Ring, a shallow mixed lake in Jutland, Denmark (55°57′51.83″N. 9°35′46.87″E) (Cuenca Cambronero, Marshall, et al., 2018). The lake was semi-pristine between the 1900s and 1950s (semi-pristine phase; SP); sewage inflow from a nearby town increased the lake’s trophic levels leading to eutrophication from the 1950s to the 1970s (eutrophic phase; EP). Sewage was diverted from the 1980s, when run-off from agricultural land surrounding the lake caused an increase in pesticides inflow (1980-1990; pesticide phase; PP). Land use declined in modern times when the lake started recovering (Recovery Phase; RP) (Cuenca Cambronero et al., 2018; Davidson et al., 2007; Eastwood et al., 2023). Four D. magna genotypes from each lake phase were used in this study for acute and chronic exposures. Before the experiments, Daphnia genotypes were acclimated at 20±1 °C, 16:8 hr light-dark photoperiod, and fed ad-libitum with 0.8mg/L of Chlorella vulgaris (strain CCAP 211/11B) for two generations to reduce interference from maternal effect and to synchronise reproduction among genotypes. The growth medium used was borehole water, collected from a deep aquifer well and showing stable physico-chemical properties tested quarterly. After purging maternal effect, clonal replicates of the 16 genotypes were exposed to phenanthrene (PHE) to test acute and chronic toxicity.
Acute toxicity exposures: Following two generations of acclimation to the experimental conditions and having controlled for maternal effect, 24-hr-old juveniles from the second or following broods were randomly assigned to experimental exposures. The OECD 202 guidelines were followed to perform immobilisation assays and extrapolate dose response curves (OECD 2004; Weber 1991). Immobilization assays are commonly used to assess the toxicity of various substances in aquatic environments. In these assays, Daphnia are exposed to different concentrations of a test substance, typically over a period of 48h. The endpoint of interest is the immobilization or lack of movement of the organisms, which serves as an indicator of toxicity. Experimental Daphnia are not fed in immobilization assays. The concentration at which a certain percentage of Daphnia become immobilized is referred to as effective concentration (EC). We conducted the immobilisation assays on 12 biological replicates of each Daphnia genotype, each including 5 individuals, across five PHE concentrations: 134 µg/L; 267 µg/L; 535 µg/L; 1069 µg/L; 2139 µg/L (N = 192). As DMSO at a final concentration of 0.001 % was used as carrier for PHE, the control used for the immobilization assays were Daphnia in borehole water (no chemicals) and Daphnia in DMSO (0.001 %). As no significant difference was observed between control and DMSO, we only include borehole water control in the analyses and plots. Immobilisation and mortality were assessed at 24h and 48h. Dose-response curves were plotted for individual genotypes and for temporal populations sampled from the four lake phases using GraphPad Prism version 10.0.3, using log-transformed immobilisation values.
Chronic toxicity exposures: The EC10 mean value across the 16 genotypes (428 µg/L of PHE) from the acute toxicity test was used to perform chronic exposures encompassing the life cycle of Daphnia. Thirteen of the 16 genotypes used in the acute toxicity tests were exposed, while LRV 0_2, LRV 13_2, and LRII 48_1 failed early in the experiment due to unforeseen circumstances and were excluded. Clonal replicates of the 13 genotypes were randomly selected from the same cultures acclimated at 20±1 °C, 16:8 hr light-dark photoperiod, and used for the immobilization. During the experiment, Daphnia cultures were fed ad-libitum with 0.8mg/L of Chlorella vulgaris (strain CCAP 211/11B).
For each genotype, six clonal replicates were exposed to 428 µg/L of PHE and six were maintained in control conditions (same as the experimental exposure minus PHE) (N = 48). During the experiment, the growth medium was replenished every other day and spiked with the same concentration of PHE at each medium change to ensure a constant exposure across the life cycle of the organisms. The experimental Daphnia were fed daily with 0.8 mg L−1 C. vulgaris and maintained at a constant temperature of 20°C. On both controls and exposed clonal replicates, a suite of fitness-linked life history traits was measured for the duration of Daphnia life cycle (i.e until each clonal replicate release their second brood). The life history traits measured were age at maturity (first time the parthenogenetic eggs are released in the brood pouch); size at maturity (distance from the head to the base of the tail spine); fecundity (number of juveniles across the first two broods); interval between broods (days between the first and second brood) and mortality. Size was measured after the release of the second brood (end of the test) using ImageJ software (https://imagej.nih.gov/ij/).