SEM-EDX analysis of heavy metals in anal papillae of Hydropsyche angustipennis larvae

Anal papillae of caddisflies are peripheral organs responsible for osmoregulation and detoxification. Investigation of morphological abnormalities in the anal papillae of Hydropsyche angustipennis ...

types of surface waters. The protection programme also applies to highly modified, polluted urban 46 streams, usually inhabited only by eurytopic species [8,9]. The wide tolerance range of these 47 organisms limits their usefulness in pollution assessment [10,11]. On the other hand, such less 48 sensitive species, resistant to substantial changes of water quality, may be used for detection of 49 dangerous pollutants e.g. heavy metals, as non-destructive environmental bioindicators [12]. 50 Evaluation of heavy metals pollution is often based on the assumption that metals can 51 accumulate in organism tissues, which reflect the environment contamination [13,14]. Such 52 bioaccumulation-based methods may also effectively track elements, which are often in 53 concentrations below detection limits [7]. However, the obtained results may not reflect the water 54 quality conditions correctly, because organisms show diverse levels of assimilation, 55 detoxification, metal tolerance and active absorption/removal of particular elements [14]. 56 Discrepancies may appear when heavy metals are being excessively assimilated from sediment 57 [15], adsorbed on the body surface [16] or accumulated in subsequent trophic levels [17]. Higher 58 levels of heavy metals concentration are observed in early larval stages, in which the presence of heavy metals in water manifest itself in their accumulation in larvae tissues as well as 73 in anal papillae abnormalities such as darkeninig and constricting [7,16,22]. It is suggested that 74 anal papillae become darkened due to accumulation of heavy metals in the epithelium. The degree 75 of such morphological irregularities in response to water pollution changes gradually, which can 76 be particularly informative [16]. 77 In our previous studies, we have also observed suggested correlation between heavy metal 78 contamination in water and tissue samples, and sublethal effects leading to morphological 79 changes in anal papillae [7,23]. However, due to small sizes of anal papillae (2-3 mm) it has not 80 been tested for heavy metal accumulation so far. In the present study, our aim was to directly 81 confirm that this particular organ (anal papillae) has an ability to accumulate heavy metals and 82 depending on the level of contamination we will be able to record progressing changes in anal 83 papillae. The method most often used for tissue analysis, such as AAS (Atomic Absorption 84 Spectrometry) was not possible to apply in this research due to small sizes of the tested organ.

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Thus, we used SEM-EDX analysis, technique enabling qualitative identification of all trace 86 elements in the tested sample, even such small sizes. By SEM-EDX microscopy analysis, we 87 were also able to compare particular parts of anal papillae e.g. pale with darkened ones. We    (Fig. 2b, 3a). In larvae with all papillae darkened completely also structural deformation 99 (rugosity/shrinking) of papillae was observed, which is the final mark of degradation (Fig. 2c).
Such changes were recorded in larvae from the L1-RC and O3-IC sampling sites. At the reference   pANOVA<0.0000) (Fig. 6). For protruded, but not darkened papillae (PALE) mean values were ten 131 times lower compared to completely darkened ones (COLRAP). In case of partially and totally 132 darkened papillae, the order of leading heavy metals was similar and as follows: Fe>Mn>Mo>Al   133 and Fe>Mo>Mn>Al, respectively. For PALE, the order was different: Al>Cu>Ni>Fe>Pb ( Fig. 6).  analysis could also indicate, point short-term discharges of sewage, which cannot always be captured by taking a water sample for analysis, as after ceasing the pollution accumulated 200 elements will be possible to trace in detoxification organs.   (Fig. 3a), then the results were averaged for individual categories of 249 the sub-lethal effects. In case of papillae classified as COLRAP and COLRAP +/-, only darkened 250 places were analyzed. The samples were analysed using always the same tilt angle -0° (approx.

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90° take-off angle) and with the accelerating voltage set to 30kV. We used 0.1% as the detection 252 limit for the EDX analysis [56]. EDX detectors collected a spectrum for every pixel of the frame.

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The spectra were then processed into a set of element intensity maps and analysed using  (Fig. 4).