Emerging contaminants and endocrine disruptors in Brazil: an overview 4th Latin American and Caribbean regional meeting on the Strategic Approach to International Chemicals Management (SAICM), Mexico City, Mexico, 19-23 August 2013 Emerging contaminants and endocrine disruptors in Brazil: an overview Wilson F. Jardim and co-workers Universidade Estadual de Campinas – UNICAMP São Paulo, Brazil wfjardim@iqm.unicamp.br

Previous and current scenario: Poor sanitation conditions; Very limited data on the presence of emerging contaminants (including EDC) in natural waters, including drinking waters; Starting in 2005 with a detailed investigation on both source and drinking waters in the State of São Paulo; 16 compounds were selected to the analyzed based upon estrogenicity, frequency of occurrence and concentration; Using a BLYES assay (luminescence) to compare the biological response to the chemical burden found in the water samples; Choosing appropriate compound that could be used as indicator of estrogenicity; In 2010 a national survey on the presence of caffeine in drinking water was carried out in 19 capitals; In 2012 the survey was repeated with 23 capitals (~ 40 million people); In vivo studies started in 2013; 25 pesticides have just being included in the list of investigated compounds;

Compounds investigated in the period 2010-2013 in drinking water Endogenous Hormones E1 E2 E3 PROG TTN Synthetics Hormones EE2 MEE NGT DES Xenostrogens ATZ nOP nNP BPA TCS PhPh CAF

Analytical Tools: LC-MS/MS YEAST BIOASSAY

Human estrogen receptor Sample with EDC Human estrogen receptor hER gene Núcleo GPD ADH1 ERE ADH1 luxC GPD luxE BLYES luxD luxA luxB frp LD – 0,09 ±0,02 ng E2/L Luminescence is measured

Pippeting of samples Pippeting of cultures Overnight incubation of strains (30 °C) Bioluminescence reading

BLYES for the 5 target compounds

Expression of results Chemical analysis ng/L for each compound BLYES ng-Equivalent of E2/L EACH COMPOUND WERE TESTED IN THE BLYES, SO IT IS POSSIBLE TO COMPARE TO MULTIPLY ITS POTENCY BY ITS CONCENTRATION AND VERIFY ITS CONTRIBUTION TO THE TOTAL POTENCY OF THE SAMPLE EXPECTED POTENCY X OBSERVED POTENCY

QUATITATIVE COMPARISION BLYES more sensitive no agreement… Expected Observed 10

Estrogenicity varies along the year dry season x wet season

IN VIVO BIOASSAYS

MATURE AND IMATURE FEMALES EXPOSED TO DRINKING WATER EXTRACTS Protocolo A – OECD 440 Protocolo B – EPA 890.1450 UTEROTHROPHIC ASSAY 21 DAYS OF LIFE SEXUAL MATURATION 21 TO 42 DAYS OF LIFE GENETIC, PHYSICAL AND MORFOLOGICAL ALTERATIONS IN: NO RESULTS, YET…..

CAMPINAS - PREVIOUS RESULTS 2010-2012 Drinking Water Surface Water 14/77 15/77 48/77 28/77 31/77 70/77 0/49 1/49 4/49 24/49 47/49

Estrogenic activity (BLYES) Blyes observed response (ngE2 equiv L-1) Caffeine (ng L-1)

PCA Samples : 103 Outliers: 1

BRAZILIAN NATIONAL CAFFEINE SCREENING SOURCE AND DRINKING WATER

BRAZILIAN REGIONS North Region Northeast Region Central Region Southeast Region South Region

caffeine screening SOUTH REGION Drinking water ng L-1 Source water ng L-1 2600 1700 2700 1300 20 25 165

caffeine screening SOUTHEAST REGION Drinking water ng L-1 Source water ng L-1 19000 40 250 1000 200 30 270 160 10 600

4 190 3 7 35 100 5 8 75 Drinking water ng L-1 caffeine screening NORTHEAST REGION

caffeine screening NORTH REGION 10 35 55 130 Drinking water ng L-1

caffeine screening CENTRAL REGION 10 600 1800 5 55 60 20 Drinking water ng L-1

Conclusions Caffeine is a resilient indicator of both treated and untreated sewage inputs and correlates not only to fecal coliforms, but also to estrogenic activity; Routine monitoring of some selected EC in both surface and drinking waters (proposed during the revision of the Brazilian Drinking Water Act); Just published the first attempt to regulate Triclosan to protect aquatic organisms in natural waters; Urgent need for biological assays to evaluate estrogenic activity both in vitro and in vivo; Which EDCs are the most important to monitor? Synergy and antagonism in real scale.

wfjardim@iqm.unicamp.br http://lqa.iqm.unicamp.br 4th Latin American and Caribbean regional meeting on the Strategic Approach to International Chemicals Management (SAICM), Mexico City, Mexico, 19-23 August 2013 Thank you! wfjardim@iqm.unicamp.br http://lqa.iqm.unicamp.br