DOI: 10.5061/dryad.51c59zwh6
title: Combined toxicity of perfluorinated compounds and microplastics on the sentinel species Daphnia magna: Implications for freshwater ecosystems

description: Persistent chemicals from industrial processes, particularly
 perfluoroalkyl substances (PFAS), have become pervasive in the environment
 due to their persistence, long half-lives, and bioaccumulative properties.
 Used globally for their thermal resistance and repellence to water and oil
 PFAS have led to widespread environmental contamination. These compounds
 pose significant health risks with exposure through food, water, and
 dermal contact. Aquatic wildlife is particularly vulnerable as water
 bodies act as major transport and transformation mediums for PFAS. Their
 co-occurrence with microplastics may intensify the impact on aquatic
 species by influencing PFAS sorption and transport Despite progress in
 understanding the occurrence and fate of PFAS and microplastics in aquatic
 ecosystems, the toxicity of PFAS mixtures and their co-occurrence with
 other high-concern compounds remains poorly understood, especially over
 organisms’ life cycles. Our study investigates the chronic toxicity of
 PFAS and microplastics on the sentinel species Daphnia, a species central
 to aquatic foodwebs and an ecotoxicology model. We examined the effects of
 perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), and
 polyethylene terephthalate microplastics (PET) both individually and in
 mixtures on Daphnia ecological endpoints. Unlike conventional studies, we
 used two Daphnia genotypes with distinct histories of chemical exposure.
 This approach revealed that PFAS and microplastics cause developmental
 failures, delayed sexual maturity and reduced somatic growth, with
 historical exposure to environmental pollution reducing tolerance to these
 persistent chemicals due to cumulative fitness costs. We also observed
 that the combined effect of the persistent chemicals analysed was 59%
 additive and 41% synergistic, whereas no antagonistic interactions were
 observed. The genotype-specific responses observed highlight the complex
 interplay between genetic background and pollutant exposure, emphasizing
 the importance of incorporating multiple genotypes in environmental risk
 assessments to more accurately predict the ecological impact of chemical
 pollutants.
description: <em>Chronic toxicity of single chemicals and mixtures
  </em> The toxicity of PFOS (70 ng/L), PFOA (7
 ng/L), PET (50 mg/L) and their two-way and three-way mixtures were tested
 on the two <em>Daphnia </em>genotypes for the duration of
 their life cycles (until they released their second brood) (Fig. 1). To
 ensure a homogeneous PET solution, a stock solution of 5g/L was prepared
 and gently stirred on a magnetic stirrer before dosing the experimental
 vials at 50 mg/L. Before the chronic toxicity test, we
 performed a qualitative assessment of <em>Daphnia</em>’s
 ingestion and egestion of microplastic over 72h. We exposed three clonal
 replicates of the two genotypes used subsequently in the chronic toxicity
 tests to 100mg/L of PET over 72h. Ingestion of microplastics was monitored
 every 24h in absence of feed. This experiment was run alongside a control
 in which <em>Daphnia </em>were fed algae. After 72h,
 <em>Daphnia </em>was transferred to clean medium every 24h, to
 visualise egestion of PET by microscope imaging. All images were captured
 using J software (Rueden et al., 2017). The medium was renewed every 24
 hours and up to 72h after the last PET feed to estimate the gut retention
 time of PET and the time to full egestion. Clonal
 replicates of the two genotypes were obtained from the stock collection of
 the University of Birmingham where they are maintained in standard
 laboratory conditions: 16:8-hr light–dark photoperiod; 0.8 mg L−1
 <em>Chlorella vulgaris</em> fed weekly; ambient temperature:
 10°C (Cuenca Cambronero et al., 2018). The growth medium used was borehole
 water, collected from a deep aquifer well and showing stable
 physico-chemical properties.<em> </em>Before commencing the
 exposures, clonal replicates of the two genotypes were acclimated in
 common garden conditions for three generations to the following conditions
 to reduce interference from maternal effects and synchronize reproduction:
 16:8-hr light–dark photoperiod; 0.8 mg L−1 <em>C.
 vulgaris</em> fed daily; ambient temperature: 20± 2 °C. After three
 generations in these conditions, 24-hr-old randomly selected juveniles
 from the second or following broods were assigned to experimental
 conditions. A total of 64 exposures were completed,
 including seven treatments and one control group for both genotypes and
 four clonal replicates per genotype and condition (Fig. 1). During the
 experiment, the growth medium (borehole water) was replenished every other
 day and spiked with the same concentration of chemical at each medium
 change to ensure a constant exposure throughout the experiment.
 Fitness-linked life history traits were measured during the experiment
 covering the <em>Daphnia</em>’s life cycle (until release of
 the second brood): 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 (Rueden et al.,
 2017).  <em>Statistical
 analyses </em> Univariate reaction norms were
 used to assess the effect of genotype (G), treatment (T -PFOS; PFOA; PET;
 PTE+PFOA; PTE+PFOS; PFOA+PFOS; PTE+PFOA+PFOS) and their interaction terms
 (G x T) on the five fitness-linked life history traits (age at maturity,
 size at maturity, fecundity, interval between broods and mortality) using
 a two-way ANOVA. A nested linear mixed effect model (LMMs) was used with
 clonal replicates as random effects nested within genotype using the
 “lmerTest” package in R version 4.3.1 (Kuznetsova et al., 2017). Before
 applying the LMMs model, the data were checked for normality using the Q-Q
 plots (residual vs. fitted value) (Zuur et al., 2010).   
 The main effects of treatment, genotype and their interactions
 were visualised using ‘reaction norm’ plots. The mortality rate per
 treatment and genotype was estimated with the survival model fit using the
 psm function in the “rms” package in R version 3.6.0 (Harrell Jr, 2023).
 The day of mortality and mortality events were combined as a response
 variable while the term “genotype” was treated as a fixed effect. The
 mortality curves per generation were plotted with the survplot function
 from the rms package in R v.6.7.1 (Harrell Jr, 2023). A separate model was
 fitted to each treatment. Multivariate effects on the
 overall fitness were calculated using multivariate statistics (MANOVA) by
 combining the life history traits (age at maturity, size at maturity,
 fecundity, and interval between broods) as response variable (y) and
 treatment and genotype as fixed terms (y ~ treatment * genotype). As per
 the ANOVA analysis of individual fitness-linked life history traits, a
 nested linear mixed effect model (LMMs) was used with clonal replicates as
 random effects nested within genotype using the “lmerTest” package in R
 version 4.3.1 (Kuznetsova et al., 2017). The multivariate reaction norms
 were visualized using phenotypic trajectory analysis (PTA) plots to
 describe the difference between control and treatments in terms of
 magnitude and direction of change following (Adams and Collyer,
 2009). <em> M</em><em>ixture
 effects</em> In the common garden experiments, we
 investigated whether the chemical mixtures (two-way and three-way
 combinations) exhibited synergistic, antagonistic, or additive effects on
 <em>Daphnia </em>ecological endpoints (fitness-linked life
 history traits). To assess this, we used a null model of additivity (Cote
 et al., 2016), which contrasts the expected additive effect of two or more
 compounds (i.e., the sum of each compound individual effects) on
 ecological endpoints with the empirical observed effect of mixtures. These
 inferences were possible because the same set of genotypes were exposed to
 the single chemicals and mixtures. The null addictive
 prediction of the joint effect of stressors combinations was calculated
 as: Emix=EA+EB+EC where EA, EB and EC are the effects
 of single compounds and Emix is the mixtures effect. For each trait ‘y’
 (age at maturity, size at maturity, fecundity, and interval between
 broods) standardized effect sizes were used corresponding to
 (y<sub>treatment</sub> - y<sub>control</sub>)/σ,
 where σ is the shared standard deviation of the pooled control and
 treatment trait values per treatment following (Cote et al., 2016). This
 analysis could not be done on mortality because individuals killed by one
 chemical cannot be killed by another simultaneously and, hence, the
 additive model cannot apply to this trait. The effect of mixtures was
 considered additive if the predicted joint effect ‘Emix’ was within the
 95% confidence intervals of the observed effect from the multiple
 stressors and not significantly different from the null model. The effect
 of multiple stressors was antagonistic if the predicted joint effect was
 significantly smaller of the null model and synergistic if the predicted
 joint effect was significantly larger than the null model. We used Welch
 Two sample t-test to assess significant departure from the null model.
 This test compares the means of the predicted null model and the empirical
 observed values for each fitness-linked life-history trait.  
description: # Combined toxicity of perfluorinated compounds and microplastics on the
 sentinel species Daphnia magna: Implications for freshwater ecosystems
 [https://doi.org/10.5061/dryad.51c59zwh6](https://doi.org/10.5061/dryad.51c59zwh6) Soltanighias et. al._metadata: fitness-linked life history traits collected following exposure to single chemicals PFOS (70 ng/L), PFOA (7 ng/L), PET (50 mg/L) and two-way and three-way mixtures of these chemicals.  Genotype ID; replicate; treatment; mortality (including mortality event and day of mortality); age at maturity; size at maturity; day of release of the first brood and number of neonates in this brood (size_1st brood); day of release of the second brood and number of neonates in this brood (size_2nd brood); overall fecundity from first and second brood; interval between broods. If mortality occurred before sexual maturity, life history traits linked to later life stages were not recorded (‘null’).