Aquatic Respiration Rate Measurements at Low Oxygen Concentrations

Despite its huge ecological importance, microbial oxygen respiration in pelagic waters is little studied, primarily due to methodological difficulties. Respiration measurements are challenging because of the required high resolution of oxygen concentration measurements. Recent improvements in oxygen sensing techniques bear great potential to overcome these limitations. Here we compare 3 different methods to measure oxygen consumption rates at low oxygen concentrations, utilizing amperometric Clark type sensors (STOX), optical sensors (optodes), and mass spectrometry in combination with 18-18O2 labeling. Oxygen concentrations and consumption rates agreed well between the different methods when applied in the same experimental setting. Oxygen consumption rates between 30 and 400 nmol L−1 h−1 were measured with high precision and relative standard errors of less than 3%. Rate detection limits in the range of 1 nmol L−1 h−1 were suitable for rate determinations in open ocean water and were lowest at the lowest applied O2 concentration

DNA damage in different Eisenia andrei coelomocytes sub-populations after in vitro exposure to hydrogen peroxide

Earthworms play an essential role in providing soil fertility and may represent an important soil contamination bio-indicator. They are able to ingest soil particles, adsorb substances throughout the intestinal epithelium into the coelomic cavity, where chemicals can come in direct contact with coelomic fluid. Earthworm coelomic fluid shelters leucocytes (coelomocytes) that differ significantly both structurally and functionally. Cellular variability could lead to different susceptibility towards contaminants possibly present in soil ecosystem. In order to define population specific dose response to chemicals and to identify a homogeneous cell population to be used as a relevant biomarker, we investigated different coelomocytes subpopulation, obtained by Percoll density gradient centrifugation (5–35 %), exposed ex vivo to H2O2 in the range of concentration 15–120 µM. DNA damage levels were assessed by the comet assay on unseparated coelomocytes and on three enriched cellular fractions (light, medium and heavy density subpopulations). All tested samples showed a dose–response genotoxic effect following H2O2 exposure. Moreover, light density sub-population appeared more susceptible to oxidative insult highlighted by a significant increase in DNA damage indexes at lower concentrations of H2O2. Present data suggested that in these experimental condition coelomocytes light fraction may represent a more sensitive biomarker of genotoxic insult

A New Highly Sensitive Method to Assess Respiration Rates and Kinetics of Natural Planktonic Communities by Use of the Switchable Trace Oxygen Sensor and Reduced Oxygen Concentrations

<div><p>Oxygen respiration rates in pelagic environments are often difficult to quantify as the resolutions of our methods for O₂ concentration determination are marginal for observing significant decreases during bottle incubations of less than 24 hours. Here we present the assessment of a new highly sensitive method, that combine Switchable Trace Oxygen (STOX) sensors and all-glass bottle incubations, where the O₂ concentration was artificially lowered. The detection limit of respiration rate by this method is inversely proportional to the O₂ concentration, down to <2 nmol L⁻¹ h⁻¹ for water with an initial O₂ concentration of 500 nmol L⁻¹. The method was tested in Danish coastal waters and in oceanic hypoxic waters. It proved to give precise measurements also with low oxygen consumption rates (∼7 nmol L⁻¹ h⁻¹), and to significantly decrease the time required for incubations (≤14 hours) compared to traditional methods. This method provides continuous real time measurements, allowing for a number of diverse possibilities, such as modeling the rate of oxygen decrease to obtain kinetic parameters. Our data revealed apparent half-saturation concentrations (K_m values) one order of magnitude lower than previously reported for marine bacteria, varying between 66 and 234 nmol L⁻¹ O₂. K_m values vary between different microbial planktonic communities, but our data show that it is possible to measure reliable respiration rates at concentrations ∼0.5–1 µmol L⁻¹ O₂ that are comparable to the ones measured at full air saturation.</p></div

Evaluation of gene expression of different molecular biomarkers of stress response as an effect of copper exposure on the earthworm eisenia andrei.

The paper reports the results of a laboratory test on the bioaccumulation and toxicological effects of sub-lethal soil concentration of copper, a widely used fungicide in organic farming, on DNA damage, a critical marker increasingly used in ecotoxicology in the earthworm Eisenia andrei. In the same experimental setting we evaluated gene expression of classical biomarker of stress induced by xenobiotic. [Heat Shock Protein 70 (HSP70) and Metallothionein (MET)], as well as genes coding for enzymes involved in detoxification of reactive oxygen species [Superoxide dismutase (SOD) and catalase (CAT)]. Additionally, expression of genes involved in the immune response were investigated: a Toll-like receptor (TLR), a receptor with cytolytic activity named Cytolytic Factor (CCF) and two antimicrobial peptides, fetidin (FET) and lysenin (LYS). Results showed significant time-dependent bioaccumulation of Cu and DNA damage at concentrations remarkably lower than those found in most agricultural soils worldwide. MET was increased as was FET and TLR. The present work gives new insights into the mechanisms of sub-lethal toxicity of copper as an environmental pollutant and in the identification of novel sub-lethal biomarkers of cellular response to the stressor such as immune response genes

Examples of modeling of oxygen depletion curves in coastal seawater samples.

<p>Oxygen depletion experiments from three incubation bottles (4, 6, 7) from St. 2 (Marselisborg Marina), September 2011. Air saturated water was injected in bottle “STOX 7” once O₂ was depleted (6 h). The dots show O₂ values recorded by the STOX sensor. Maximum rates (V_max) and half saturation constants (K_m) were determined by least squares fits (red solid line) using Eq. (2), derived from Michaelis-Menten kinetics. The method gave a relatively poor fit that might result in an underestimation of the K_m value for the “STOX 6”.</p

Time course of oxygen depletion in a STOX sensor incubation with coastal seawater.

<p>Example of STOX sensor data from incubation of water from Station 2 (Marselisborg Marina, September 2011). Only minimum and maximum readings during each cycle are plotted and connected with lines. The difference between maximum and minimum readings is used as a measure of the O₂ concentration. The initial amplitude of the signal (11 pA) corresponds to an O₂ concentration in the bottle of 200 nmol L⁻¹. Injections of air saturated water were made at 1 h (2 mL, ∼600 nmol L⁻¹), and at 6.7 h (4 mL, ∼1200 nmol L⁻¹), as showed by the red line.</p

Overview of measured community respiration rates.

<p>Summary of average V_max (nmol L⁻¹ O₂ h⁻¹), K_m (nmol L⁻¹ O₂) ± standard error, number of replicates in brackets, for St. 1 and 2 in both summer and autumn. Incubation temperatures in the laboratory are reported. R² values assess the fit between the data and the Michaelis-Menten and Jassby (Italic) models used to estimate the apparent V_max or CR rates, and K_m values of the communities. St. 2* refers to V_max and K_m values estimated with data recorded after injection of air saturated water, during the second progress curve in the same incubation.</p

Results from the control experiment (Exp. 1).

<p>Four different replicates with different oxygen concentrations are shown. A 64 h 1 mL of air saturated 0.05 M HCl in Milli-Q water, corresponding to 244 nmol L⁻¹ O₂, was added for sensor calibration.</p

Comparison of CR rates measure with the STOX method and with Winkler titrations.

<p>Time course of oxygen depletion during incubations (A, C, E, G) and respiration rates at different O₂ concentrations (B, D, F, H) measured by the STOX method (from 0 to 2 µmol L⁻¹, white bars), and by Winkler titrations (at air saturation, grey bars). A, B: St. 1 (Randers Fjord); C, D: St. 2 (Marselisborg Marina) at concentrations of 0–2 µmol L⁻¹ and E, F: Marselisborg Marina at concentrations of 0–1 µmol L⁻¹. G, H: St. 3 (Hanstholm). Rates are averages ± standard error (n = 3). Both Michaelis-Menten kinetic parameters and Jassby and Platt ones (in parenthesis) are presented.</p