DICLOFENAC: NEW DATA ON CHRONIC TOXICITY AND BIOCONCENTRATION IN FISH

Diclofenac (DCF) is a widely used nonsteroidal anti-inflammatory drug that is regularly detected in surface waters. To support a robust aquatic risk assessment, two early life stage (ELS) tests, compliant with the Organisation for Economic Co-operation and Development (OECD) test guideline 210, were conducted in rainbow trout and in zebrafish. Population relevant endpoints, such as hatching, growth, and survival, and in the trout study, histopathological effects in potential target organs, were examined. The bioconcentration of DCF in rainbow trout was measured in a separate study according to OECD test guideline 305. The bioconcentration factor (BCF) in rainbow trout remained below 10, demonstrating no relevant bioconcentration of DCF in fish. In the rainbow trout ELS test, the no observed effect concentration (NOEC) including histopathology was 320 µg/L. The effect of DCF on zebrafish growth was less clear, and the NOEC can be interpreted as 10 µg/L. However, for a number of reasons, the authors consider the moderately reduced growth of zebrafish exposed to concentrations of up to 320 µg/L not a repeatable, treatment-related effect of DCF. This leads us to a conclusion that DCF has, with high probability, no adverse effect on both fish species up to 320 µg/L. This NOEC indicates a sufficient safety margin for fish populations, because concentrations of DCF in European rivers are in the range of ng/L to low µg/L. Environ. Toxicol. Chem. 2013;32:442–452. © 2013 SETAC


Instrumental setup for total radioactivity
Liquid scintillation counters Packard TRI-CARB 2500 TR and 2900 TR equipped with DPM and luminescence options.
All measurements were performed after scintillation background correction and all samples were determined at least in duplicate. The following scintillation fluids and reagents were used: A Irga-

Lipid measurement in fish
The fish pools were homogenized in a Waring Blendor in the presence of dry-ice, and the homogenized tissue samples were dried with Hydromatrix drying reagent. Thereafter, the homogenates were extracted in two cycles in the ASE (Accelerated Solvent Extraction) system using hexane. The hexane solvent extracts were evaporated under as gentle steam of nitrogen.
The lipid pellets were dried at 105°C to a constant weight.

Calculation of BCF's
The steady state BCF SS is the ratio of the concentration in fish (C f ) at the plateau level (i.e. at the last three successive fish sampling dates) to the measured concentration in water (C w ). The kinetic bioconcentration factor (BCF K ) was calculated by fitting the uptake rate constant k 1 and the depuration rate constant k 2 by the non-linear parameter estimation program Origin (MicroCal). All bioconcentration factors were based on the concentration of total radioactivity in parent equivalents in fish (μg equivalents/g fish) and on the average concentration of total radioactivity (C w ) during exposure.

Histopathological evaluation in the ELS test with rainbow trout
Twenty fish (five per tank replicate) from each test concentration and from the control were randomly selected for histopathological examination of the liver, kidney and gills at the end of the exposure period. These organs were examined for any lesion or alteration.