In vitro determination of the endocrine disrupting capacity of microplastics
Abstract
Since the development of plastic in the early 20th century, it has become essential in day-to-day living. The demand for plastic has grown exponentially to a point where there are over 300 million tonnes of plastic produced globally every year. It is estimated that 60–80% of all man-made litter consists of plastic and in South Africa, that amounts to approximately 630 000 tonnes of which more than 90 000 tonnes end up in South Africa’s oceans. Microplastics (MPs) in particular pose a hazard to any environment that they are found in. Not only are they inhaled, ingested by, or attached to aquatic and terrestrial organisms, they also aid in the transport and redistribution of harmful organisms such as bacteria and chemicals in the environment. There are two ways in which chemicals pose a problem: firstly by the adsorption and desorption of harmful chemicals such as persistent organic pollutants and polyaromatic hydrocarbons to the MPs in the environment. The MPs facilitate the distribution of these harmful compounds. Secondly, the leaching from plastics of harmful additives added during production such as phthalates and bisphenol A. Some of these additives and chemicals are known to cause adverse effects in organisms including endocrine disruption.
Polyvinyl chloride (PVC) is one of the most widely used plastics currently in production. However, its use has been under constant inquiry due to the harmful chemicals used in its production. Furthermore, PVC has a low recyclability and is easily degraded weathering. This leads to the abundance of PVC MPs that are found in all environments. This study aimed to determine the endocrine disrupting capacity of virgin and recycled PVC leachates.
In this study, virgin, recycled and unrecycled ground polyvinyl chloride MP pellets were leached under environmentally relevant conditions to simulate natural leaching. The leachates were assessed for endocrine disrupting potential using in vitro reporter gene assays. The human breast cancer cell lines, MDA-kb2 with endogenous androgen receptors (AR) and glucocorticoid receptors (GR), and T47D-KBluc with endogenous oestrogen receptors (ER) were used to determine the effects. These cells are genetically modified to include the firefly luciferase reporter gene which is coded downstream of the promoter, producing luciferase upon activation of the receptor. The luciferase catalyses a light-producing reaction and therefore the receptor-binding activity of the leachates could be quantified, as the amount of light produced is directly proportional to receptor binding.
The PVC MP pellets were leached in ethanol at 4°C and 30°C for 24, 48, and 96 h respectively. They were exposed to cells to determine AR, GR, and ER activation, and both AR and ER inhibition using testosterone, dexamethasone, oestradiol, flutamide, and ICI-1782,780 as positive
controls, respectively. Furthermore, cytotoxicity was tested as an endpoint using the human duodenum adenocarcinoma HuTu-80 cell line and performing an MTT viability assay.
None of the leachates activated the AR or GR. Likewise, no inhbition of both the AR and ER was recorded. There was however, evidence of competition for binding to ER. The ER activation assay was not successful due to a high background activation of the assay. The cytotoxicity data proved that the content of the leachates changes over time with the PVC being leached for 24 and 48 h being more cytotoxic than those leached for 96 h. Furthermore, the recycled plastic was more cytotoxic than the unrecycled plastic in this leaching conditions, suggesting that additives used in the recycling process may be cytotoxic.
The lack of endocrine disruption within the experimental conditions of this study only accounts for a small amount of research that needs to be done to properly assess the effects of microplastics in the environment. The in vitro determining of the endocrine disrupting capacity of microplastic is an excellent tier 1 screening method. However, conclusions cannot be made regarding MP leachates without further chemical analysis of the mixtures, specific mixture testing and in vivo tests, and wider range of leaching variables.