The zebra mussel is no ordinary freshwater mollusc. Classified among the top 100 most harmful invasive species, it inflicts costly biofouling damage on industrial infrastructure worldwide, clogging pipes, screens and cooling systems wherever it establishes populations. Its native range spans eastern Bulgaria, southern Romania and Ukraine, where it inhabits temperate lakes, rivers and reservoirs along the Golyama Kamchia River system. The species lives an average of three to five years, though some specimens survive up to fifteen, and individuals grow from roughly one to 1.6 centimetres in their first year, with a maximum total length of about five centimetres. Reproduction, development and growth depend on a delicate balance of water temperature, pH, calcium concentration, dissolved oxygen, turbidity and salinity, with breeding occurring at water temperatures between 12 and 28 degrees Celsius.

Calcium stands at the heart of shell construction, and the zebra mussel demands more of it than most bivalves. According to the researchers, adult specimens require calcium concentrations between 12 and 15 milligrams per litre to grow and survive. Values below 15 milligrams per litre signal a risk to reproduction and growth, while concentrations of 15 to 30 milligrams per litre are considered moderate and 30 to 80 milligrams per litre high. Yet while several earlier studies documented element concentrations in the soft tissues of the species, analyses of the shell itself remained scarce. This gap motivated the present investigation, which sought to determine the chemical composition of shells from mussels in their native areal and to correlate those findings with the elemental content of the surrounding water and sediments.

The PIXE technique offered the analytical muscle to do this. The method is highly sensitive to elements with atomic numbers above 10, thanks to the thin beryllium window of the detector most commonly employed. It is non-destructive, meaning samples are preserved for further analysis, and it enables the examination of large numbers of small-mass samples in short time, with irradiation of a single sample lasting less than one minute. Its sensitivity is an order of magnitude higher than comparable methods, and the detection limit for elements in biological samples falls below one milligram per gram, benefiting even trace element detection. PIXE has found applications across chemistry, medicine, biology, archaeology, agriculture, materials science, geology, environmental research and pollution monitoring, and has previously been used to determine elemental composition in the bodies of various aquatic inhabitants.

Sampling took place in the spring of 2009 at the Ticha Dam, built along the Golyama Kamchia River approximately 18 kilometres from the town of Varbitsa and 40 kilometres from Shumen. With a water volume of 311,103 cubic metres and a circumference exceeding 100 kilometres, it is the dam with the largest circumference in Bulgaria. Water temperature at collection ranged from 6 to 9 degrees Celsius. The mussels were sorted into three size groups: small shells up to one centimetre, medium shells between one and two centimetres, and large shells above two centimetres. Water samples were taken from 20 centimetres below the surface and sediments from the uppermost layer. All samples were dried to constant weight, finely ground into powder with a porcelain mortar, and pressed into 13-millimetre pellets using a five-tonne hydraulic press. The pellets were coated with a thin carbon layer to make them electrically conductive and dissipate accumulated charge during measurement.

The irradiation itself was carried out at the PIXE camera of the 2.5 MV AN2000 Van de Graaff accelerator at the National Laboratories of Legnaro, part of the National Institute of Nuclear Physics in Italy. Shell samples were bombarded with a proton beam of 1.8 MeV energy and 4-millimetre diameter. Ionisation of the atoms in the samples resulted from irradiation with these accelerated protons, and the characteristic X-ray emission was detected by a high-purity germanium detector with an energy resolution of 160 eV at the 5.89 keV manganese K-line. The accelerator current was kept below 5 nanoamperes, with total charge deposited on targets on the order of 2 microcoulombs. Spectra were processed with the GUPIX WIN program, and calibration relied on standards of known elemental content prepared from the International Atomic Energy Agency’s Soil-7 reference material under geometry similar to that of the samples.

To quantify the relationship between shell chemistry and environmental conditions, the team calculated the Metal Pollution Index, or MPI, a geometric mean of metal concentrations in each sample. Values above 1 indicate a relationship between chemical elements and the environment, while values below 1 suggest no such relationship. The results were unambiguous. The MPI values for small, medium and large shells were 0.61, 2.86 and 4.13 respectively, demonstrating a strong environmental dependence during the mussel’s growth and development. As the mussels grew, they accumulated chemical elements from their surroundings in their shells, with the accumulation intensifying dramatically across ontogenetic stages.

The element-specific results revealed striking patterns. Calcium, potassium, iron and manganese were detected in all shell samples. Calcium appeared in approximately the same amount across all three size groups, within experimental error, confirmed by a coefficient of variation of 18.43 percent. Potassium concentrations differed across all three samples, with the highest values in the largest mussels and the lowest in medium-sized ones, yielding a coefficient of variation of 108.06 percent for the shells. Iron told an even more dramatic story: a significantly higher amount of 13 milligrams per gram was measured in the shells of the largest mussels, compared with 1.2 milligrams per gram in smaller and 1.7 milligrams per gram in medium representatives, producing a coefficient of variation of 125.91 percent. Manganese followed the same trend, with a coefficient of variation of 134.07 percent. Titanium, chlorine and zinc appeared in only two of the three groups, with titanium detected in medium and large specimens at twice the concentration in the largest mussels compared with the medium ones. Chromium showed the lowest coefficient of variation of all, at 6.73 percent.

Multivariate statistical analysis using IBM SPSS Statistics 19 brought the elemental relationships into sharp focus. A Pearson test across nine variables produced 36 correlation coefficients, of which 15 were negative and 21 positive. Potassium correlated positively with all other elements, with its highest coefficients against titanium at 0.704 and iron at 0.689. The highest positive correlation of all appeared between sulphur and chlorine at 0.911. Calcium, by contrast, correlated positively only with sulphur and chlorine, and negatively with all other elements, with its largest negative correlations against titanium at -0.932 and iron at -0.929, the very elements most strongly correlated with potassium. The team concluded that iron, titanium and potassium share the same sources, accumulating in the shells from the water and sediments of the mussel’s habitat. Cluster analysis confirmed these relationships, grouping potassium, manganese, iron, titanium and chromium together as the elements most interconnected and most strongly linearly correlated with one another, falling into the shells and influencing growth as concentrations changed with shell size.

The growth-rate findings may be the most consequential of all. The tendency of shell growth was associated with an increasing iron-to-calcium ratio: 0.0041 in small shells, 0.0043 in medium shells and 0.043 in large shells, a tenfold increase from juvenile to adult. The manganese-to-calcium ratio followed a similar trajectory, rising from 0.0003 in small mussels to 0.004 in large ones. Iron in the shells accumulated mainly from water, with a correlation coefficient of 0.872, while manganese accumulated from both water and sediments, with correlation coefficients of 0.9845 and 0.9541 respectively. The correlation between iron and manganese in the shells themselves reached 0.9998. The researchers propose that the accumulation of manganese, and especially iron, is crucial for the development of the species, and that the absence of these two metals in a habitat may affect populations and limit the ability of mussels to reach the reproductive ontogenetic stage. The iron concentration measured in adult mussels, 10.3 micrograms per gram, proved comparable to that of 72 marine bivalve species, though younger specimens deviated significantly despite high iron levels in the surrounding water and superficial sediment layers. The team notes that the accumulation of these elements in the individual white and black stripes of the zebra-like shell pattern would be of considerable interest, but that this forms the topic of their next study. For now, the work provides valuable baseline data on the shell chemistry of the species in its native range, information that may ultimately inform methods for mitigating its impact as an invasive species far beyond Bulgarian waters.

Subject of Research: The application of PIXE analysis in the identification of key elements for the ontogenetic development of Zebra mussels in their native areal

Article Title: The application of PIXE analysis in the identification of key elements for the ontogenetic development of Zebra mussels in their native areal

Article References: Arhangelova, N., Salim, S., Valkanov, K., Kolev, N., Marinova, P., Veleva, L., & Natchev, N. (2026). The application of PIXE analysis in the identification of key elements for the ontogenetic development of Zebra mussels in their native areal. One Ecosystem, 11, Article e167875. https://doi.org/10.3897/oneeco.11.e167875

Image Credits: AI Generated

DOI: 10.3897/oneeco.11.e167875

Keywords: zebra mussel, PIXE, Dreissena polymorpha, invasive species, elemental analysis, freshwater ecology, bioaccumulation, application, analysis, identification, elements, ontogenetic