The Muck that Can Be the Testing of Toxicity to Soil Microbes A recent publication in a high-profile journal described a novel technique to measure toxicity of chemicals to soil microorganisms (article #1). In principle, this is a great and innovative idea. But in both the telling and in the verification the great idea lost its luster.

Scientists from Duke University developed a microbial assay to test whether or not different chemicals impact microbial functions. The assay is focused on Paracoccus denitrificans. This is an organism that transforms soil nitrate to nitrogen gas in a process that is referred to as denitrification. The assay measures actually denitrification of added NH3 along with gene expression and cell viability.

First, let me rant a little bit about the telling. The abstract starts off with language filled with opinion and prejudice. For example, “…still little is known regarding the ecological impacts of non-regulated contaminants found in biosolids.” The authors go onto say that biosolids are unregulated regarding organic contaminants and that the contaminants that are “likely to accumulate in biosolids” likely have certain characteristics that likely make them “particularly persistent and bio-accumulative” and that they “pose a potential risk to the human food chain.” This language may get you published, but it is increasingly difficult to take, particularly when in doing so the authors ignore the wealth of research and literature on the topic that suggests just the opposite. I have several decades of NBMA libraries to back me up here.

So let’s go from my ranting to a discussion of the actual paper which, in light of the other papers in the library, raises serious questions about the applicability of the Duke University approach.

The Duke authors focused on anti-microbial compounds in their assessment of this new method. They included triclocarban (TCC) and triclosan (TCS) along with other types of antimicrobials. They added compounds at a range of 3 log units to experimental vessels that also contained glucose, bacterial cells and NO2-. I was not clear reading the article what the final concentrations of the compounds in the vessels were. My first recalculation of their discussion came up with 0.03, 0.3 and 3 ppm TCS, which is well above what you would see in solution in a biosolids amended soil. But let’s say that the calculations are realistic. The authors saw reduced denitrification at the two highest levels of TCC and TCS addition and so concluded that these were environmentally relevant toxicity indicators that showed that biosolids-borne TCC and TCS were hazardous.

However, the other indicators that they used, gene expression and cell viability, told a different story. Gene expression saw stimulation in comparison to the control for all genes measured at the lowest level of compound addition, with decreases at the highest level for some of the genes measured. Cell viability told a similar story, with increases at the lowest level of compound addition in comparison to the control. To me this says that this is not a perfect assay. To the authors, it says that these two indicators are not “as sensitive” as the denitrification portion of the assay.

Concerns about the assay are not the main point. The kicker here is that the method does not take into account the importance of the matrix in the determination of the toxicological effect. What that means is that biosolids carrying TCC or TCS can have an enormous impact on the availabilities and toxicities of these compounds. If you are familiar with biosolids, you remember this whole concept from the discrepancy in toxicity with metals added to soils as salts and metals added to soils in a biosolids matrix.

The remainder of the library is devoted to the impact of the matrix on the toxicity of TCC and TCS to soil microbes as well as the impact of biosolids application to the soil microbial community.

The 2nd article in the library is about the toxicity of ‘biosolids- borne TCC’ to terrestrial organisms, including worms and soil microorganisms. In this article, two biosolids were added to soils, containing 24 and 7 ppm TCC. The authors used standard EPA methods to look at CO2 respiration and the complete N cycle, including transformation of organic N to NH4, and NH4 to NO3 for biosolids added to a sandy soil. To set up their study, the authors first did a lab test to see what type of concentrations would be appropriate to include and found some repression of denitrication when biosolids that had TCC concentrations of 34 ppm were added to the soil at 22 metric tons per hectare. For the final test they spiked biosolids up to 73 ppm TCC and also included a treatment with 717 ppm biosolids to represent a 30 year loading case. The biosolids were added to the sandy soil at 22 metric tons per hectare. The authros saw a spike in CO2 emissions for all rates of TCC addition, likely related to increased microbial activity from the biosolids addition. They also saw no impact on NH4 or NO3 concentrations as a result of the TCC. The authors noted that the bacteria responsible for these transformations are likely not as sensitive to TCC. They also noted that concentrations of NO3 decreased by the end of the study for all treatments and suggested that this might be the result of localized denitrification reactions. It is important to note here that TCC degrades very slowly in soils with a half-life measured in many years rather than days or months.

The 3rd article in the library conducted the exact same study, but focused on TCS instead of TCC. Here the authors observed a no-effect soil TCS concentration of 10 ppm. That would mean a biosolids concentration of 2000 ppm for a single application at agronomic rates. Previous studies have shown a relatively rapid degradation rate for biosolids-borne TCS, with a half-life measured in weeks rather than months. Both the 2nd and the 3rd article are from George O’Connor’s lab at the University of Florida.

It is interesting to note that the authors of the first study did reference these two articles at the very end of their study. Their comment was that “it might be advisable to expand this method to more complex media that better simulate biosolids and soil.”

The Duke authors did not reference the last two articles in the library. These articles used sophisticated microbial techniques to identify changes in the soil microbial community following biosolids application.

The 4th article looked as soil bacterial and archaea populations following three years of biosolids or synthetic fertilizer application to corn. Both bacteria and archaea transform ammonia to nitrate in a process that is called nitrification. The authors found that rates of nitrification increased in the biosolids amended soils. These soils also had higher amounts of the genes from both the archaea and bacteria that are responsible for this process.

The last article in the study looked at the soil microbial community following more than 15 years of biosolids applications to dryland wheat. Biosolids are applied every 4 years, with a wheat crop grown every two years in a wheat fallow rotation. The soils for the microbial analysis were sampled 2 years after the last biosolids application and so represent the long term effects of biosolids on these soils. The authors saw increased total microbial biomass in the biosolids amended soil in comparison to the fertilizer control. This biomass consisted of higher numbers of both gram positive and negative bacteria as well as anaerobic bacteria in comparison to the control treatments.

Interesting to note, the authors of the Duke study referenced neither of these two studies, both of which suggest that biosolids addition is beneficial to soil microorganisms rather than detrimental.

Sally Brown