Phthalate pollution in an Amazonian rainforest
Phthalates are ubiquitous contaminants and endocrine-disrupting chemicals that can become trapped in the cuticles of insects, including ants which were recognized as good bioindicators for such pollution. Because phthalates have been noted in developed countries and because they also have been found in the Arctic, a region isolated from direct anthropogenic influence, we hypothesized that they are widespread. So, we looked for their presence on the cuticle of ants gathered from isolated areas of the Amazonian rainforest and along an anthropogenic gradient of pollution (rainforest vs. road sides vs. cities in French Guiana). Phthalate pollution (mainly di(2-ethylhexyl) phthalate (DEHP)) was higher on ants gathered in cities and along road sides than on those collected in the pristine rainforest, indicating that it follows a human-mediated gradient of disturbance related to the use of plastics and many other products that contain phthalates in urban zones. Their presence varied with the ant species; the cuticle of Solenopsis saevissima traps higher amount of phthalates than that of compared species. However, the presence of phthalates in isolated areas of pristine rainforests suggests that they are associated both with atmospheric particles and in gaseous form and are transported over long distances by wind, resulting in a worldwide diffusion. These findings suggest that there is no such thing as a “pristine” zone.
KeywordsPhthalates Pollution Tropical rainforests Ants DEHP
Of all of the pollutants found across the globe, phthalates (mainly di(2-ethylhexyl) phthalate (DEHP)) are some of the most widely distributed. Phthalate esters are used in many industrially made products, such as cosmetics, pesticide carriers, insect repellents, vinyl, cables, tubing, films, paints, adhesives, PVC, and inks. They are also used as plasticizers (i.e., to make plastics more flexible). Because phthalate esters do not chemically bind to plastic polymers, they migrate to the surface of the polymer matrix where they may more easily leach into the air, water, or food. They have been detected in the air (including in aerosols), water, soil, different sediments, and animal tissue, including that of humans (Teil et al. 2006; Alves et al. 2007; Babich and Osterhout 2010; Williams et al. 2010; Gaudin et al. 2011; Salapasidou et al. 2011; Choi et al. 2012; Huang et al. 2013).
Hundreds of scientific papers and many newspaper articles have chronicled the effects of endocrine-disrupting chemicals (EDCs, mainly phthalates, and bisphenol A), which have been associated with human pathologies (e.g., negative effects on the male reproductive tract, breast and testicular cancers, disruption of the neuroendocrine system, allergies, and asthma) (Saillenfait and Laudet-Hesbert 2005a, b; Desdoits-Lethimonier et al. 2012; Manzetti et al. 2014). Moreover, we know that the toxicity of certain pollutants is greater than previously thought and frequently results in transgenerational effects (e.g., in fish; Schwindt et al. 2014). Furthermore, the impact can be exacerbated by interactions between contaminants or “cocktail effects” (e.g., pesticide combinations on bees) (Vidau et al. 2011; Gill et al. 2012) or between contaminants and natural stressors, including malnutrition, osmotic perturbations, and global warming (Rhind 2009; Holmstrup et al. 2010).
Phthalate air pollution has both acute and chronic effects ranging from minor upper respiratory irritations to chronic respiratory and heart diseases, lung cancer, acute respiratory infections in children, and chronic bronchitis in adults. In addition, short- and long-term exposure to phthalate pollution has also been linked to premature mortality and reduced life expectancy (Kampa and Castanas 2008) and transgenerational effects through epigenetic mechanisms (Doyle et al. 2013; Manikkam et al. 2013; Rissman and Adli 2014). Many reports have indicated that the phthalates found in dust in houses are associated with asthma and allergies in both children and adults (Ait Bamai et al. 2014).
Phthalates have been found on insect cuticles such as those of ants, crickets, and honey bees, something which has been taken as evidence of their ubiquity (Cavill and Houghton 1974; Kather et al. 2011; Lenoir et al. 2012); they can also become trapped in the wax of honey bee combs (Gómez-Ramos et al. 2016). DEHP and dibutyl phthalate (DBP) are toxic at high doses for Folsomia candida springtails, causing modifications in symmetry (Jensen et al. 2001; Kristensen et al. 2004). Phthalates deposited in large quantities on Lasius niger ant cuticle remained in dead, control individuals, while they were adsorbed and metabolized in less than 5 days and so returned to their basic level, in live individuals (Lenoir et al. 2014). At doses corresponding to chronic exposure levels, phthalates reduce ant queen fecundity and stimulate an immune response in workers (Cuvillier-Hot et al. 2014).
Because phthalates are transported everywhere in the atmosphere above developed countries (Choi et al. 2012; Blanchard et al. 2013) and because they have been found in the Arctic (Xie et al. 2007), a region isolated from direct anthropogenic influences, they appear to be widespread. To verify this, we hypothesized that their presence in isolated pristine Amazonian rainforests would provide strong evidence that the planet’s atmosphere is thoroughly polluted by these compounds.
Ants are present everywhere, are found in almost every part of the food web, and constitute the most abundant animal taxon in tropical ecosystems (Longino et al. 2014; see also Basset et al. 2015 for tropical insect diversity). Consequently, ants represent important bioindicators based on the degree to which they have been contaminated by pollution. So, we compared the phthalate pollution levels of ants from isolated pristine rainforest in French Guiana, far from any human activity, with areas having increasing levels of anthropogenic perturbation, including urban areas, where plastics and many products containing phthalates (e.g., detergents, building materials, and furniture) are in constant use. However, because phthalates are rapidly degraded by microbial activity and abiotic processes (i.e., hydrolysis, photocatalytic oxidation, and photolysis) (Staples et al. 1997; Zhou et al. 2005; Yuan et al. 2010; Huang et al. 2013; Manzetti et al. 2014), the levels recorded are likely much lower than those associated with the original source of contamination. We also aimed to identify the various phthalates present because, due to concerns over their safety, the most frequently used (i.e., DBP, diisobutyl phthalate (DiBP), and DEHP) are progressively replaced by heavier molecules, which have already been found in soft plastics produced in Asia (Barušić et al. 2015; AL, personal observation).
Materials and methods
Ants were captured with metal forceps and placed directly into glass vials containing hexane; they were never in contact with plastics and were left in the vials until the analyses were run. At that point, they were removed from the vials, and the solvent evaporated. Then, the extract was redissolved in 10 μL of hexane to which 2 μL of hexane containing 400 ng of eicosane (C20) was added as an internal standard (we verified that all the hexane used was phthalate free). We injected 2 μL of each redissolved extract into a Perkin-Meyer gas chromatograph-mass spectrometer (GC-MS) functioning at 70 eV and with a source temperature of 230 °C. The GC-MS was equipped with a ZB-5HT column (30-m L × 0.25-mm ID × 0.252 μm df; 5 % phenyl—95 % dimethylpolysiloxane). The following temperature program was used: 2 min at 80 °C, increased by 10 °C/min to reach 320 °C, and a 10-min hold at 320 °C (for a total of 36 min). An external mixture of phthalates is generally used to quantify phthalate acid esters (PAEs) (Teil et al. 2006). Eicosane is frequently used as the standard in hydrocarbon analyses, so we utilized it here to compare this study with previous ones (Lenoir et al. 2012, 2014; Cuvillier-Hot et al. 2014). We used ion 149, typical of phthalates, as the basis for our analyses of the phthalate peaks (Cao 2008; Valton et al. 2014; Barušić et al. 2015). This method is less sensitive but much more effective in differentiating phthalates from other hydrocarbons, particularly DEHP from 5MeC25 (Lenoir et al. 2014). We calculated the quantity of each compound relative to the eicosane internal standard. The threshold for DEHP quantification is 0.20 ng, so that, for small ants, we placed five workers in the extract vial. We analyzed a total of 243 samples.
Since the species ranged in size, the results were normalized and presented in terms of nanogram per milligram of dry weight (DW), as in Lenoir et al. (2014).
Data are presented as means ± standard errors (SE), and statistical analyses were conducted using ANOVAs and the Newman-Keuls post hoc test for multiple comparisons (R software).
Results and discussion
The different phthalates recorded
Different phthalates found on ants in French Guiana for Solenopsis and all other ant species (mean ng/mg DW ± SE, % of samples containing phthalates, % quantities related to the total amount of phthalates)
Phthalates (ng/mg DW)
We also found on Guianese ant cuticules two new phthalates, di(2-ethylhexyl) terephthalate ((DEHTP) = dioctylterephthalate (DOTP)) and diisononyl phthalate 35 isomers (DINP), which are recently being used instead of DEHP (Rastogi 1998; Abe et al. 2012). DEHTP can be passively transferred by simple contact between ants and fragments of plastic children’s toys (A. Lenoir, unpublished results), explaining why it occurred on urban Guianese ants. DINP was detected in 22.7 % of Solenopsis and 31.8 % of other ants gathered around the Nouragues research station and in the cities of Cayenne (at the harbor), Kourou, and Sinnamary; it was also noted at the Petit-Saut field station and along the road to the dam. When present, DINP only represented 1 to 2 % of the phthalates. In the Nouragues research station, it was recorded in the pieces of flagging tape tied around trees to delimit parcels. DINP is found in toys, childcare products, PVC, flagging tape, and many soft plastics (Barušić et al. 2015). Its metabolites have been detected in human urine across the globe (Saravanabhavan 2012), and although it seems to be less toxic than the more common phthalates (Babich and Osterhout 2010), it was placed on California’s official list of carcinogens (Tomar et al. 2013).
The plastic tubing used to delimit parcels at the Nouragues research station contains BBP and DEHP in small quantities, likely explaining their presence on ants. However, these compounds were also noted on ants gathered far from any human activity, such as the top of the inselberg.
Anthropogenic gradient of pollution
The cuticular phthalate levels observed for urban Guianese ants are similar to those noted for the ant L. niger in Europe (i.e., 2 ng/ant fresh weight, corresponding to 5 ng/mg DW) (Lenoir et al. 2012). Yet, a perfect comparison would require using the same species.
Phthalates were ubiquitous around the Nouragues research station, as they were found in ants from the camp, the forest, and the top of the inselberg. The levels were low, ranging from 0.5 (the top of the inselberg) to 2 ng/mg DW, and did not differ significantly between sites (p = 0.06, but near significance for the top of the inselberg, p = 0.055), so that human activity in and around the station is not likely responsible for the phthalate pollution noted deep in the rainforest and on the top of the inselberg.
Therefore, our hypothesis that phthalate pollution is globally ubiquitous is likely confirmed as, in addition to their presence in the Arctic (Xie et al. 2007), we found them in other areas isolated from direct anthropogenic influence, including parts of the Amazonian rainforest and the top of an inselberg. These results strongly suggest that contaminants arrive from the atmosphere both with air particles and in gaseous form (see Blanchard et al. 2014; Cecinato et al. 2012; Gao and Wen 2016; Teil et al. 2016; Xie et al. 2005). For example, in the Paris region, phthalate pollution ranges from 10 to 100 ng m−3 of total air and 80 % in the gaseous phase. It is more concentrated in urban areas compared to forest sites (Teil et al. 2016).
Variation in phthalate levels across ant genera
In conclusion, it appears that phthalates are universal contaminants and are probably major constituents of generalized anthropogenic pollution, which is a leading cause of human health problems. They may also be playing a role in the mass extinctions of the Anthropocene, which are affecting both vertebrate and, albeit less visibly, invertebrates (Dirzo et al. 2014). Phthalates are the major pollutants disseminated throughout the world in gaseous form and on atmosphere particles (Teil et al. 2016). Our results show that they are found in different levels on ant cuticle based on a gradient of urbanization, so ants can be considered good bioindicators due to their ubiquity and ease of sampling them. It is thus imperative to continue to study the pollution of ant populations, most particularly in tropical rainforests.
Financial support for this study was provided by a CNRS/Centre d’Études de la Biodiversité Amazonienne (CEBA) project entitled “Phthalate pollution in an Amazonian rainforest” (PPAR). We are grateful to Chloé Fasilleau and Chloé Moyse (École Polytechnique, Université de Tours, France) for the analysis of the data, to Jessica Pearce-Duvet and Andrea Yockey-Dejean for proofreading the manuscript, and to Jacques H. C. Delabie (Laboratório de Mirmecologia, CRC, Ilhéus, Bahia, Brazil) for the identification of the ants. We would like to thank the staff of the CNRS Nouragues research station and the Laboratoire Environnement de Petit-Saut for furnishing logistical assistance.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interest.
- Barušić L, Galić A, Bošnir J, Baričević L, Mandić-Andačić I, Krivohlavek A, Mojsović Ćuić A, Đikić D (2015) Phthalate in children’s toys and childcare articles in Croatia. Curr Sci 109:1480–1486Google Scholar
- Huang J, Nkrumah PN, Li Y, Appiah-Sefah G (2013) Chemical behavior of phthalates under abiotic conditions in landfills. Rev Environ Contam Toxicol 224:39–52Google Scholar
- Saravanabhavan, GMJ (2012) Human biological monitoring of diisononyl phthalate and diisodecyl phthalate: a review. J Environ Pub Health 2012:ID 810501Google Scholar
- Schwindt AR, Winkelman DL, Keteles K, Murphy M, Vajda AM (2014) An environmental oestrogen disrupts fish population dynamics through direct and transgenerational effects on survival and fecundity. J Appl Ecol 51:582–591. doi:10.1111/1365-2664.12237
- Tomar RS, Budroe JD, Cendak R (2013) Evidence on the carcinogenicity of the diisononyl phthalate (DINP). California Environmental Protection Agency. http://oehha.ca.gov/prop65/hazard_ident/pdf_zip/DINP_HID100413.pdf