DOI: 10.5061/dryad.kh18932fr
title: Interactions between phenanthrene exposure and historical chemical stress: Implications for fitness and ecological resilience of the sentinel species Daphnia magna

description: 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. 
description: Study system: The <em>Daphnia</em>
 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
 <em>D. magna </em>genotypes from each lake phase were used in
 this study for acute and chronic exposures. Before the experiments,
 <em>Daphnia</em> genotypes were acclimated at 20±1 °C, 16:8 hr
 light-dark photoperiod, and fed ad-libitum with 0.8mg/L of
 <em>Chlorella vulgaris</em> (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,
 <em>Daphnia</em> 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 <em>Daphnia</em> are
 not fed in immobilization assays. The concentration at which a certain
 percentage of <em>Daphnia</em> become immobilized is referred
 to as effective concentration (EC). We conducted the immobilisation assays
 on 12 biological replicates of each <em>Daphnia</em> 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 <em>Daphnia</em> in borehole
 water (no chemicals) and <em>Daphnia</em> 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 <em>Daphnia</em>. 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, <em>Daphnia</em> cultures were fed
 ad-libitum with 0.8mg/L of <em>Chlorella vulgaris</em> (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 <em>Daphnia</em> 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/).
description: 1. 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. 2. 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