Sewage sludge toxicity assessment using earthworm Eisenia fetida: can biochemical and histopathological analysis provide fast and accurate insight?

Sewage sludge (SS) is a complex organic by-product of wastewater treatment plants. Deposition of large amounts of SS can increase the risk of soil contamination. Therefore, there is an increasing need for fast and accurate assessment of SS toxic potential. Toxic effects of SS were tested on earthworm Eisenia fetida tissue, at the subcellular and biochemical level. Earthworms were exposed to depot sludge (DS) concentration ratio of 30 or 70 %, to undiluted and to 100 and 10 times diluted active sludge (AS). The exposure to DS lasted for 24/48 h (acute exposure), 96 h (semi-acute exposure) and 7/14/28 days (sub-chronic exposure) and 48 h for AS. Toxic effects were tested by the measurements of multixenobiotic resistance mechanism (MXR) activity and lipid peroxidation levels, as well as the observation of morphological alterations and behavioural changes. Biochemical markers confirmed the presence of MXR inhibitors in the tested AS and DS and highlighted the presence of SS-induced oxidative stress. The MXR inhibition and thiobarbituric acid reactive substance (TBARS) concentration in the whole earthworm's body were higher after the exposition to lower concentration of the DS. Furthermore, histopathological changes revealed damage to earthworm body wall tissue layers as well as to the epithelial and chloragogen cells in the typhlosole region. These changes were proportional to SS concentration in tested soils and to exposure duration. Obtained results may contribute to the understanding of SS-induced toxic effects on terrestrial invertebrates exposed through soil contact and to identify defence mechanisms of earthworms.

Repeated sampling of Atlantic cod (Gadus morhua) for monitoring of nondestructive parameters during exposure to a synthetic produced water.

The past decades of monitoring discharges from oil and gas industry have revealed that although there are indications of adverse effects in tissues of aquatic organisms, little is known about their temporal development. Furthermore, observations in wild-caught individuals have not been clearly reproduced in laboratory studies or caging studies, and vice versa, and the results are therefore not easily interpretable. There is clearly a need for exposure studies designed for monitoring the development of effect markers in individual fish over chronic periods to low contaminant levels. Through repetitive nondestructive sampling, the progression of effects may be monitored in individuals, significantly reducing the number of fish needed in exposure studies. A laboratory exposure study was designed to be able to monitor selected parameters in individual Atlantic cod (Gadus morhua). Passive integrated transponders in combination with visible implant elastomers were used to study individual fish during the exposure period (44 wk). Fish were measured (weight and length) and a blood sample was taken for analysis of hematocrit, DNA damage (micronucleus), and oxidative stress (total oxyradical scavenging capacity) at up to seven time points. There were no apparent adverse effects of treatments on the health of experimental fish, frequency of micronucleated erythrocytes, or oxidative stress in whole blood. It is possible that the time scale was not sufficient for development and detection of parameters included here or that red blood cells may not be a suitable matrix for the selected analyses. Future studies need to include other parameters in blood to investigate their sensitivity to low-concentration exposures.

Detection of DNA damage in haemocytes of zebra mussel using comet assay.

The aim of the study was to use the comet assay on haemocytes of freshwater mussel, Dreissena polymorpha Pallas, for detection of possible DNA damage after exposure to pentachlorophenol (PCP) and to evaluate the potential application of the comet assay on mussel haemocytes for genotoxicity monitoring of freshwater environment. Zebra mussels were exposed for seven days to different concentrations (10, 80, 100, 150 microg/l) of PCP and in the river Sava downstream from Zagreb municipal wastewater outlet. Significant increase in DNA damage was observed after exposure to PCP at doses of 80 microg/l and higher and after in situ exposure in the river Sava as well. This study confirmed that the comet assay applied on zebra mussel haemocytes may be a useful tool in determining the potential genotoxicity of water pollutants.

Increase in number and size of kidney concretions as a result of PCP exposure in the freshwater snail Planorbarius corneus (Gastropoda, Pulmonata).

Molluscan kidneys are able to excrete solids in the urine in the form of concretions. It is thought that increased formation of these concretions occur under pollutant, environmentally or reproductive induced stress. This study examined the formation of concretions in the kidney of the freshwater snail Planorbarius corneus L. experimentally exposed to pentachlorophenol (PCP). Light microscopic histopathological analysis of the PCP-exposed P. corneus revealed significantly enhanced production of the kidney concretions when compared to the kidneys of control individuals. Measurements of the number of kidney concretions, the apparent area of the concretions, and the epithelial area filled with concretions indicated an increase in the number and size of concretions in all treated snails. Lipofuscin content of excretory cell concretions was detected.

Detection of micronuclei in haemocytes of zebra mussel and great ramshorn snail exposed to pentachlorophenol.

The frequency of micronuclei (MN) induced by pentachlorophenol (PCP) in haemocytes of zebra mussel, Dreissena polymorpha Pall. and great ramshorn snail, Planorbarius corneus L. was determined over a 14 days of exposure (sampling after 4, 7 and 14 days) under laboratory conditions. PCP doses for zebra mussel ranged from 10 to 150 microg/l, and for ramshorn snail from 10 to 450 microg/l. Micronuclei were detected after bisbenzimide fluorescent staining. Positive responses were observed in both species. The mean MN frequencies in treated mussels ranged between 0.69 and 7.50 per thousand, and between 2.07 and 13.80 per thousand in treated snails. The spontaneous MN levels in mussels averaged from 0.5 to 2.75 per thousand, and in snails from 1.56 to 2.00 per thousand. Our results suggest that haemolymph of both species represent an appropriate test tissue in environmental genotoxicity assessment.