The Study

Sourcing the Menu: Water & Fertilizers: Ubiquitous Contamination Sources

NOTE: This is an edited version of portions of Appendices 3 of the “Revised Stealth Syndromes Study Protocol as approved by the University of California San Francisco Medical School Committee on Human Research.

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Irrigation water: farm ponds, biosolids and recycled municipal wastewater.

With rare exceptions, irrigation involves plastic pipes, tanks, tubing, drip devices, and valves which leach varying amounts of plastic chemical contaminants.

Well water may be the purest source, but is less available as water tables drop from drought and over-pumping. The distribution lines of a well are usually plastic.

Municipal water may be the second best source depending upon the treatment methods, filtering and added chemicals. However, municipal water distribution is increasingly using plastic pipes for mains and secondary distribution lines.

Farm ponds are questionable for direct use because of wildlife, livestock and other uses in the upstream drainage area.

In addition to BPA and phthalates, recycled municipal wastewater contains scores of harmful chemicals including pharmaceuticals, illegal narcotics and other undesirable compounds that can find their way into animal flesh and meat.

Reviews of the use of recycled municipal wastewater indicate that contaminates remain in the water and can be absorbed the tissues of growing plants.

Significantly, commercial hay fields are frequently fertilized to increase production. That fertilizer may be a commercial product, or biosolids — sludge from sewage treatment plants which frequently contain heavy metals.

Assessment of endocrine disruption and oxidative potential of bisphenol-A, triclosan, nonylphenol, diethylhexyl phthalate, galaxolide, and carbamazepine, common contaminants of municipal biosolids


  • Relative potency determined for six individual contaminants in a suite of bioassays
  • Multiple-modes of action of individual contaminants demonstrated in different in vitro assays
  • Comprehensive assessment of potential in vitro effects associated with carbamazepine


The use of biosolids as a soil conditioner and fertiliser is hindered by the limited knowledge on the risks of micro-contaminants they contain. This study investigated the binding of six organic contaminants commonly found in biosolids, to the estrogen (ER), androgen (AR), aryl hydrocarbon (AhR), and transthyretin (TTR) receptors and their redox activity. Triclosan (TCS), bisphenol-A (BPA), and technical nonylphenol (TNP) had affinity for the TTR with relative potencies of 0.3, 0.03, and 0.076 respectively. Further, binding to TTR was the only toxicological response observed for carbamazepine, which induced sub-maximal response and relative potency of 0.0017. Estrogenic activity was induced by BPA, galaxolide (HHCB), diethylhexyl phthalate (DEHP) and TNP with BPA having the strongest potency of 5.1 × 10−6 relative to estradiol. Only BPA showed androgenic activity but it was not quantifiable. BPA also showed anti-androgenic activity along with TCS, HHCB, and TNP in the order of TNP > HHCB > TCS ~ BPA (relative potencies 0.126, 0.042, 0.032, 0.03). No compounds exhibited anti-estrogenic or AhR activity, or were redox-active in the dithiothreitol assay. The results highlight the multiple modes of action through which these compounds may impact exposed organisms, and the concentrations at which effects may occur. This allows assessment of the likelihood of effects being observed at environmental concentrations, and the potential contribution of these compounds.

Biosolid Risks

Unfortunately, papers in the last couple of years are pointing toward biosolids as a source of microplastics, drug-resistant bacterial and genes, as well as artificial estrogens and other endocrine disruptors.

The safe and environmentally responsible use of biosolids is a goal worth striving for. However, biosolids are not — yet — ready for prime time. Below is a small sample of recent published studies which point to the need for more caution and the need for further research to protect the public health.

Contamination by E. Coli and other pathogens

Rainfall-runoff of anthropogenic waste indicators from agricultural fields applied with municipal biosolids


  • Biosolids-derived emerging contaminants found in simulated rainfall runoff.
  • Biosolids are a potential source of anthropogenic contaminants to surface waters.
  • Runoff contaminant concentrations relatively constant across multiple rain events.
  • Intense and frequent precipitation dramatically increases contaminant load.
  • Some contaminants undergo in situ attenuation unrelated to precipitation events.


The presence of anthropogenic contaminants such as antimicrobials, flame-retardants, and plasticizers in runoff from agricultural fields applied with municipal biosolids may pose a potential threat to the environment. This study assesses the potential for rainfall-induced runoff of 69 anthropogenic waste indicators (AWIs), widely found in household and industrial products, from biosolids amended field plots. The agricultural field containing the test plots was treated with biosolids for the first time immediately prior to this study. AWIs present in soil and biosolids were isolated by continuous liquid-liquid extraction and analyzed by full-scan gas chromatography/mass spectrometry. Results for 18 AWIs were not evaluated due to their presence in field blank QC samples, and another 34 did not have sufficient detection frequency in samples to analyze trends in data. A total of 17 AWIs, including 4-nonylphenol, triclosan, and tris(2-butoxyethyl)phosphate, were present in runoff with acceptable data quality and frequency for subsequent interpretation. Runoff samples were collected 5 days prior to and 1, 9, and 35 days after biosolids application. Of the 17 AWIs considered, 14 were not detected in pre-application samples, or their concentrations were much smaller than in the sample collected one day after application. A range of trends was observed for individual AWI concentrations (typically from 0.1 to 10 μg/L) over the course of the study, depending on the combination of partitioning and degradation mechanisms affecting each compound most strongly. Overall, these results indicate that rainfall can mobilize anthropogenic contaminants from biosolids-amended agricultural fields, directly to surface waters and redistribute them to terrestrial sites away from the point of application via runoff. For 14 of 17 compounds examined, the potential for runoff remobilization during rainstorms persists even after three 100-year rainstorm-equivalent simulations and the passage of a month.

Antibiotic Resistance

Novel Antibiotic Resistance Determinants From Agricultural Soil Exposed To Antibiotics Widely Used In Human Medicine And Animal Farming


Antibiotic resistance has emerged globally as one of the biggest threats to human and animal health. Although the excessive use of antibiotics is recognized for accelerating the selection for resistance, there is a growing body of evidence suggesting that natural environments are “hotspots” for the development of both ancient and contemporary resistance mechanisms. Given that pharmaceuticals can be entrained onto agricultural land through anthropogenic activities, this could be a potential driver for the emergence and dissemination of resistance in soil bacteria. Using functional metagenomics, we interrogated the “resistome” of bacterial communities found in a collection of Canadian agricultural soil, some of which had been receiving antibiotics widely used in human medicine (macrolides) or food animal production (sulfamethazine, chlortetracycline and tylosin) for up to 16 years. Of the 34 new antibiotic resistance genes (ARGs) recovered, the majority were predicted to encode for (multi)drug efflux systems, while a few share little to no homology with established resistance determinants. We characterized several novel gene products, including putative enzymes that can confer high-level resistance against aminoglycosides, sulfonamides, and broad range of beta-lactams, with respect to their resistance mechanisms and clinical significance. By coupling high-resolution proteomics analysis with functional metagenomics, we discovered an unusual peptide, PPPAZI 4, encoded within an alternative open-reading frame not predicted by bioinformatics tools. Expression of the proline-rich PPPAZI 4 can promote resistance against different macrolides but not other ribosomal-targeting antibiotics, implicating a new macrolide-specific resistance mechanism that could be fundamentally linked to the evolutionary design of this peptide.


Antibiotic resistance is a clinical phenomenon with an evolutionary link to the microbial pangenome. Genes and protogenes encoding for specialized and potential resistance mechanisms are abundant in natural environments, but understanding of their identity and genomic context remain limited. Our discovery of several previously-unknown antibiotic resistance genes from uncultured soil microorganisms indicates that soil is a significant reservoir of resistance determinants, which, once acquired and “re-purposed” by pathogenic bacteria, can have serious impacts on therapeutic outcomes. This study provides valuable insights into the diversity and identity of resistance within the soil microbiome. The finding of a novel peptide-mediated resistance mechanism involving an unpredicted gene product also highlights the usefulness of integrating proteomics analysis into metagenomics-driven gene discovery.