Comparison between droplet digital PCR and reverse transcription-quantitative PCR methods to measure ecotoxicology biomarkers.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is currently the gold-standard technique for detecting and quantifying messenger RNA. However, without proper validation, the method may produce artefactual and non-reproducible cycle threshold values generating poor-quality data. The newer droplet digital PCR (ddPCR) method allows for the absolute quantification of targeted nucleic acids providing more sensitive and accurate measurements without requiring external standards. This study compared these two PCR-based methods to measure the expression of well-documented genes used in ecotoxicology studies. We exposed Mediterranean mussels (Mytilus galloprovincialis) to copper and analyzed gene expression in gills and digestive glands using RT-qPCR and ddPCR assays. A step-by-step methodology to optimize and compare the two technologies is described. After ten-fold serial complementary DNA dilution, both RT-qPCR and ddPCR exhibited comparable linearity and efficiency and produced statistically similar results. We conclude that ddPCR is a suitable method to assess gene expression in an ecotoxicological context. However, RT-qPCR has a shorter processing time and remains more cost-effective.

Ecological impacts of long-term application of biosolids to a radiata pine plantation.

Assessment of the ecological impact of applying biosolids is important for determining both the risks and benefits. This study investigated the impact on soil physical, chemical and biological properties, tree nutrition and growth of long-term biosolids applications to a radiata pine (Pinus radiata D. Don) plantation growing on a Sandy Raw Soil in New Zealand. Biosolids were applied to the trial site every 3 years from tree age 6 to 19 years at three application rates: 0 (Control), 300 (Standard) and 600 (High) kg nitrogen (N) ha(-1), equivalent to 0, 3 and 6 Mg ha(-1) of dry biosolids, respectively. Tree nutrition status and growth have been monitored annually. Soil samples were collected 13 years after the first biosolids application to assess the soil properties and functioning. Both the Standard and High biosolids treatments significantly increased soil (0-50 cm depth) total carbon (C), N, and phosphorus (P), Olsen P and cation exchange capacity (CEC), reduced soil pH, but had no significant effects on soil (0-20 cm depth) physical properties including bulk density, total porosity and unsaturated hydraulic conductivity. The High biosolids treatment also increased concentrations of soil total cadmium (Cd), chromium (Cr), copper (Cu) and lead (Pb) at 25-50 cm depth, but these concentrations were still considered very low for a soil. Ecotoxicological assessment showed no significant adverse effects of biosolids application on either the reproduction of springtails (Folsomia candida) or substrate utilisation ability of the soil microbial community, indicating no negative ecological impact of bisolids-derived heavy metals or triclosan. This study demonstrated that repeated application of biosolids to a plantation forest on a poor sandy soil could significantly improve soil fertility, tree nutrition and pine productivity. However, the long-term fate of biosolids-derived N, P and litter-retained heavy metals needs to be further monitored in the receiving environment.