Effect of phthalates on development, reproduction, fat metabolism and lifespan in Daphnia magna
Graphical abstract
Introduction
Phthalates, or, phthalic acid esters (PAEs) are commonly used as plasticizers for polyvinyl chloride (PVC) to increase polymer's flexibility, softness and durability. Phthalates are used as solvents in various industrial and consumer products including toys, medical devices, building materials, automobile parts, electrical cables, and food packaging (Chen et al., 2008; Larsson et al., 2017). The global production of phthalates has reached approximately 6–8 million tons per year and consumption in Europe is approximately 1 million tons per year (Dobrzynska, 2016; Mackintosh et al., 2006; Norrgren et al., 1999). Since phthalates are not chemically bonded to PVC they leach out and enter into the environment (Gao and Wen, 2016). Di-(2-ethyl hexyl) phthalate (DEHP), diethyl phthalate (DEP) and dibutyl phthalate (DBP) are among the most commonly detected phthalates in aquatic environments (Li et al., 2016; Selvaraj et al., 2015). These three phthalates represents 90% of phthalates detected in the Kaveri River water in India with total concentration ranging from 313 to 4640 ng/L with DEHP constituting 57% followed by DEP (22%) and DBP (11%) (Selvaraj et al., 2015). While the maximum allowable concentration of DEHP in drinking water according to WHO guideline is 8 μg/L, the maximum allowable concentration in inland and other surface waters according to USEPA and EU directive is 1.3 μg/L (DIRECTIVE_2013/39/EU, 2013; USEPA, 2012; WHO, 2003). However, the widespread use of phthalates has resulted to its detection in many countries well above the limit. Measured phthalates concentration in the environment range from 0.1–370 μg/L in surface water and 0.1 ng/g–100 μg/g for river sediments (Bono-Blay et al., 2012; Sha et al., 2007; Sung et al., 2003).
The regulations passed so far for different phthalates only restricts their application in specific consumer products. DEHP, the most abundant and widely used phthalate, was the first to be restricted in Europe in 1999 in toys and other childcare articles that can come in direct contact to children's mouth (1999/815/EC, 1999). 2007 EU's REACH regulation banned five more phthalates (DBP, BBP, DINP, DIDP and DNOP) in toys and childcare articles that are available above 0.1% concentration (EUREACH_ANNEX_XVII, 2007). Restriction of the aforementioned phthalates, more specifically DEHP, has led to the increased use of alternatives such as non-restricted phthalates DEP and other non-phthalate compounds (Larsson et al., 2017). DEP, one of the most used phthalates in personal care products is used as a solvent in creams, lotions, baby shampoos and perfumes, results in high levels of infant exposure and has been detected in urine samples of infants (Sathyanarayana et al., 2008). DEP is generally characterized as a non-endocrine active compound with low toxicity (Heindel et al., 1989; Howdeshell et al., 2008) however, recent studies have shown DEP also has adverse effects on humans and animals (Harley et al., 2017; Pradhan et al., 2017; Zhou and Flaws, 2017).
Human and animal studies have shown that phthalates have adverse effects on reproductive organ development. In male rats, DEHP and DBP disrupt fetal testicular testosterone synthesis (Hannas et al., 2011; Parks et al., 2000). The average human exposure is estimated to be between 2.32 and 12 μg/kg/d for DEP, 0.84–5.22 μg/kg/d for DBP and 0.71–4.6 μg/kg/d for DEHP based on urinary biomarker studies from Germany and USA populations (Koch and Calafat, 2009).
Organisms in direct contact with the aquatic system are especially vulnerable to phthalate exposure. Studies have shown that phthalates have various biological effects on aquatic organisms. In crustaceans, decreased locomotor activity and survival has been reported for Gammarus pulex (Thuren and Woin, 1991). In Artemia salina phthalates leads to decreased hatching success (Sugawara, 1974). In fish phthalates can lead to abnormalities in development and reproduction (Carnevali et al., 2010; Sun and Liu, 2017). In lower organisms like protozoans Tetrahymena pyriformis (Jaworska et al., 1995) and algae (Acey et al., 1987) phthalates show negative effect on growth.
Daphnia magna is a well-established in vivo model in toxicological studies, and is the most commonly used system for ecotoxicological testing worldwide (Jordao et al., 2016). D. magna is considered an important species in ecological food webs, as they are principal grazer of algae, bacteria and protozoans and a primary forage for fish (Colbourne et al., 2011). The use of D. magna can help to predict toxicity of phthalate to the aquatic ecosystem (Poynton et al., 2007; Tessier et al., 2000).
Organisms respond to environmental contaminants and stressors by altering their gene expression profiles as a direct or indirect result of exposure (Steinberg et al., 2008). Understanding gene expression analysis associated to physiological and phenotypic alterations can provide further insight into the molecular mechanisms of toxicity. However, most previous studies with D. magna and other aquatic model systems have not explored the mechanism of phthalates toxicity in detail. Studies have mainly been based on phenotypic changes, survival, locomotion and reproduction success and little is known about the effects at the molecular level. Hence, it is important to develop sensitive methods based on gene expression profiles and link this to different physiological end points to better understand the molecular mechanisms. In this study we have used gene expression and physiological end points including reproduction, lifespan, metabolism, hatching, developmental abnormality and survival to compare the toxicity of three phthalates (DEHP, DEP and DBP) and also to make a thorough investigation of phthalates mechanism of action.
The main objective of the present study was to analyze the toxicity of the restricted phthalates DEHP and DBP as well as the non-restricted phthalate DEP and to understand the mechanisms of action using the aquatic crustacean, D. magna in the laboratory under controlled conditions. Our study indicates that phthalates can have negative effect on development, reproduction, fat metabolism and lifespan. The detrimental effect was not only observed for the banned phthalate DEHP, but the substitute phthalates (DEP and DBP) are equally toxic to D. magna.
Section snippets
Chemicals
The phthalate compounds used in this study includes DEP - CAS No. 84-66-2, DBP - CAS No. 84-74-2 and DEHP - CAS No. 117-81-7. The compounds were purchased (Sigma) with stated purities in excess of 99%. To obtain experimental exposure concentration the phthalates were dissolved in dimethyl sulfoxide (DMSO; Sigma). The final assay concentration of DMSO was 0.1%.
D. magna culture, maintenance and exposure
D. magna from Daphtoxkit (MicroBioTests Inc., Belgium) were used to study the effect of phthalates. Ephippia were activated by rinsing in
Phthalates affect development and induce stress response genes
In order to determine the effect of three phthalates, DEHP, DEP and DBP (Fig. 1) in D. magna, survival assay was performed to evaluate if the selected concentrations (1 and 10 μM) of the three phthalates can induce acute toxicity. Exposure of D. magna for 48 h after hatching and 96 h from the egg stage did not result in any lethality (data not shown). Other studies including Brown et al. (1998) and Scholz (1994) also did not find any acute toxicity of DEHP in D. magna. DEHP also does not show
Conclusion
The use of phthalates have increased over the years and consequently their levels are increasing in water bodies. This could lead to serious negative effects on aquatic life if the use of phthalates are not regulated. In this study we have analyzed three phthalates in freshwater organism D. magna using different physiological end points to understand phthalates toxicity as well as mechanisms of action. Except the DEHP mediated reproduction increase, most of the other results obtained in this
Acknowledgements
This study was financed by the Knowledge Foundation, Sweden (20150084) and Örebro University.
We would like to thank Prof. Per-Erik Olsson for the critical reading of the manuscript.
Conflict of interest
There are no conflicts of interest.
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