Strategies for Monitoring of Endocrine Disrupting Chemicals in Aquatic Environment
Sammanfattning: The protein biomarker vitellogenin in fish is discussed alongside alterative strategies for monitoring of endocrine disrupting chemicals. The first objective was to find a cheap, robust and generic method for the quantification of vitellogenin. A new sample preparation method was developed for the indirect quantification of vitellogenin through alkali-labile phosphate (ALP) with final analysis using spectrophotometry. An improved method with molybdate as colour reagent had a detection limit of 3.2µg phosphate / ml plasma, which was further improved using molybdate in combination with malachite green giving a detection limit of 0.3µg / ml. The first method was applied for measurement of background levels of ALP in male and female crucian carp (Carassius carassius), as well as in in fish 7 days after intraperitonal injection of 17β-estradiol. The malachite method was applied for ALP measurement in juvenile rainbow trout (Oncorhynchus mykiss) from a fish farm which were placed in flow-through tanks at three locations along the brook of Vallkärra, Lund, Sweden. This brook is contaminated with leachate water from a covered landfill. In comparison to reference fish, ALP was significantly higher (66%) in fish placed close to the landfill. With the ALP method based on malachite green the obstacle of insufficient detection limit has been overcome enabling very wide usage of ALP measurements in the future. A miniaturized microporous membrane, liquid-liquid extraction was developed using a hollow fibre for analysis of steroid hormones during controlled exposure studies with fish or analysis in sewage water. The sample preparation was combined with gas chromatography and a detection limit of 1.6, 3, and 10 ng/L was achieved for 17β-estradiol, estrone and 17α-ethinylestradiol respectively. This method is simple, environmentally friendly and omits the use of additional sample clean-up despite measuring in such a complicated matrix as sewage. For improved risk assessment a method for chemical measurement of bioavailability of persistent organic pollutants with endocrine effects was developed. With selective supercritical fluid extraction it was possible to estimate and predict how large fraction of PCBs in a contaminated soil that was bioavailable to earthworms. By confirming the uptake in worms it was found that the extraction procedure could give a 93% accurate estimation of bioavailability.
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