Accumulation of microplastics in Zebra mussel (Dreissena polymorpha, Pallas, 1771) in the sand pit lake Kazichene, Bulgaria

In recent years, microplastic contamination has received worldwide distribution and specific attention. As a result, detecting sensitive bioindicators is crucial to establish the pollution. The aim of the present study is to investigate the accumulation of microplastics in zebra mussels (Dreissena polymorpha, Pallas, 1771) from the sand pit lake Kazichene. The natural range of the zebra mussel in Bulgaria includes the Danube River delta, the Black Sea rivers and coastal lakes. However, it is considered a wide spread invasive species to inland freshwaters. The use of invasive species as bioindicators over native species is advisable in order to protect the local biodiversity. In total 20 mussels of D. polymorpha were used for the analysis. The biological tissues of the mussels were digested with the aid of 30% H2O2 at 65°C for 24 - 48 hours. Microplastics (772 pcs. in total) were detected in all zebra mussel samples, indicating microplastic contamination in Lake Kazichene. Three main shapes have been identified: fragments, fibres and pellets, with the highest proportion of the fragments. The colours of the microplastic particles found were black, red, yellow and transparent, dominated by those of black colour, followed by red. The length of the fibres found in the zebra mussels ranged from 0.25 to 5 mm. There was no strong correlation between the body length/meat weight of zebra mussels with the degree of microplastic accumulation in the mussels.


Introduction
In recent years, microplastics have received widespread attention as one of the most persistent environmental pollutants.In 2004, Professor Thompson from the UK first proposed the term microplastics to address the issue of plastic pollution [1].In most current studies, microplastics are defined by the size of plastic particles, meaning particles smaller than 5 millimeters are considered microplastics [2].Microplastics have many different types, and according to their shape, they can be divided into four main types: fibers, fragments, pellets, and films [3].Other authors divided microplastics into five categories according to their shapes: fragments, fibers, beads, foam and pellets [4].
Investigating microplastic contamination in organisms can be used to understand whether they are contaminated by microplastics, to explore the source and mode of contamination, etc., and to analyze the possibility that the pollution may be passed on to other organisms through the food chain which 1305 (2024) 012005 IOP Publishing doi:10.1088/1755-1315/1305/1/012005 2 could lead to more serious impacts.Bivalves contaminated with microplastics filtered from the water column may affect the dynamics of microplastic availability from planktonic to benthic compartments through particle elimination from feces or pseudofeces [5,6].Once ingested microplastics may undergo biofragmentation turning into smaller particles disposed of through feces [7,8], thus making these particles available to smaller detritivorous hydrobionts [6].On the other hand, a certain aquatic organism can be used as an indicator for monitoring of microplastic pollution in the aquatic environment, and the characteristics of microplastics in the organism can be used to indirectly reflect the microplastic pollution in the aquatic environment in which it lives [1].
There is a lot of research on microplastics in organisms, but it mainly focuses on the study of marine species.It is estimated that 267 species worldwide are affected by plastic waste [9].The research on freshwater organisms mainly focuses on freshwater fish, while there are few reports on gastropods and bivalves.It is proved in laboratory conditions that dreissenids ingest microplastics [10,11].Pastorino et al. [12] investigated microplastics in zebra mussels in Lake Iseo, Italy.The authors measured and compared the microplastic levels in D. polymorpha sampled from sites at different distances from the wastewater treatment plant (WWTP) to determine whether it can be used as a bioindicator of microplastic pollution in lakes.Hoellein et al. [13] studied microplastics in dreissenid mussels from Milwaukee River and Lake Michigan -United States.
Previous studies on microplastic pollution in Bulgaria have primarily concentrated on the Black Sea and coastal lakes, with no prior research conducted on freshwater bodies [14,15,16,17].Therefore, it is important to investigate the microplastic pollution in Bulgaria's freshwater bodies.Zebra mussels can be a biological indicator of microplastic pollution in freshwater because the species has worldwide distribution.As filter feeders, mussels are suitable objects for studying aquatic pollution, and as an invasive species the zebra mussel, supports large populations and is suitable to reduce the use of native species for such type of studies [12].
This article aimed to study the accumulation of microplastics in Zebra mussels (D. polymorpha) from Lake Kazichene, Bulgaria.The work hypothesis was that bigger individuals would accumulate more significant amount of microplastic particles.

Sampling area
For this study samples collected from Lake Kazichene (figure 1) were used.Samples were collected in July 2022.The lake is located in the western part of the village of Kazichene near Sofia City -Bulgaria.The sampling point was situated in South -East part of the Lake.The lake is no longer used for sand and gravel extraction and has a maximum depth of 12 m and an elevation of 551 m.The bottom substrate in the lake is mostly sand, gravel and stones in the shallow part, and silt-clay and sandy in the deeper part.Zebra mussels were found attached to the stems of aquatic plants and to a concrete structure at a depth of about 1 m [18].On the sand pit Lake Kazichene's territory there is a hydro park for water sports such as wakeboarding and water skiing.
The coordinates of the sampling point are: N: 42.657200; E: 023.449340 (figure 1).

Sample collection and primary processing of biological samples
Adult zebra mussels were sampled by the use of hand bottom drag with an entrance of 17.5 cm, a fine scraping edge, and a handle length of 1.70 m [19].Samples were taken from the littoral part of the lake, at about 1 to 2 meters underwater.After collection the mussels were washed with water from the lake on site, and then transported to the laboratory packed with aluminum foil in a cooling bag at 4 ℃.In the laboratory the samples were stored in a freezer at -20 ℃ before analysis.
Frozen zebra mussels were thawed in laboratory conditions.Twenty mussels with unharmed shells and no external signs of injury were selected.They were washed with distilled water to remove contaminants on the surface of the mussels, thus avoiding the possibility of microplastic particles from the external environment entering the samples prior to the digestion.Each zebra mussel was numbered, and then the length, width, and height of the shell of each individual were measured to the nearest 0.01 mm using a digital caliper.The mussel flesh was then dissected and weighed using an analytical balance to the nearest 0.0001 g. Figure 1.Sampling point at sand pit lake Kazichene and position of the Lake near Sofia city and on the map of Bulgaria.

Digestion of biological tissues:
After weighing, the soft tissues were rinsed with distilled water to avoid secondary contamination from the air.They were placed in beaker cups previously rinsed with distilled water and numbered with the code of the respective zebra mussel.To each sample, 20 ml of 30% H2O2 was added.The beakers were covered with aluminum foil to avoid contamination with microplastics from the air and prevent hydrogen peroxide's evaporation.One hydrogen peroxide control was added under the same conditions but without mussel tissue.All beakers were placed in a thermostat at 65°C for 24-48 hours [4,20,21].Larger mussels took longer to digest.When the solution was clear and there was no visible tissue debris and gas bubbles, degradation of the biological tissue was considered complete.Once the degradation process was complete, the beakers were removed from the thermostat, allowed to cool, and then proceeded to filter the samples.A glass vacuum filtration system and glass-fiber microfilters (Whatman GF/C; pore 1.2 μm; diam.47 mm) were used.After filtration was complete, each microfilter was stored in a closed glass Petri dish until the visual examination.

Contamination control during the experiment
Strict control measures were taken during the experiment to prevent contamination of the samples with microplastics from the laboratory environment.Prior to the experiment, the hydrogen peroxide was filtered through a glass-fiber filter to exclude contamination of the reagent with microplastics.Two controls were placed, one to assess airborne contamination during the experiment and another to control contamination in the experimental environment.All laboratory supplies used in the experiments were made of non-plastic materials.All glassware and forceps were rinsed 3 times with distilled water before use.The beakers in which the biological tissue was placed for digestion were covered with aluminum foil to avoid contamination with microplastics from the air.A filter moistened with distilled water was placed in an open Petri dish to control airborne contamination.Cotton laboratory coats and nitrile gloves were worn during the experiments, and doors and windows were closed to reduce air movement and strictly control potential contamination, during the experimental process.Each sample was evaluated by two researchers.If the results did not match, a third evaluation was performed and the two closest values were used.

Visual identification of the microplastics in the samples
All the filters were examined under a stereomicroscope at a magnification of 6 to 60 times.Microplastics were identified by shape and color, counted and photographed.During identification, the entire filter was viewed in the shape of the letter "Z" from left to right to reduce errors [22].
The microplastic particles detected were recorded in a protocol and classified into the following categories based on their morphological characteristics: pellets, fragments, fibers, beads, and foam [4].Only the fiber length (mm) was measured with an eyepiece-micrometer.
As the colors have direct effects on the photoaging of plastics as well as they can affect the colonization of microorganisms on microplastics, the adsorption, release, and degradation of pollutants associated with microplastics, and the biological toxicity of microplastics [23], it is very important to study this indicator.The colors of the microplastics were recorded as a single color or as transparent.If the particle was both transparent and colored, the color was recorded.

Data processing and analysis
Microplastics abundance, the relationship between mussel biometrics, and microplastic content were determined using linear regression analysis.Correlation analysis was used to test the relationship between microplastic abundance and mussel characteristics.For the data procession Microsoft Office Excel 2016 was used.

Ethical statement
Permission for the field study conducted on public land was not provided because the study was conducted on the sand pit Lake Kazichene, which is no longer in use for sand and gravel extraction.The subject of the study was the Zebra mussels (D. polymorpha) which is an invasive species for the lake.No rare or protected species were threatened during the field sampling.

Results and discussion
Microplastics were found in all samples of zebra mussels -772 pcs. in total, which indicates that Lake Kazichene is contaminated by microplastics.The average amount of microplastics in zebra mussels was 38.6 pieces per mussel.Zebra mussels accumulate microplastics in their bodies by filtering the water and accidentally ingesting suspended microplastics.When the ratio between microplastics and zooplankton in the aquatic environment is 2.73, this can lead to confusion, leading to accidental ingestion of microplastics [24], which then induce bioaccumulation effects by enhancing transfer along the food chain [25,26].In this study, the type of microplastic in the zebra mussel was mainly fragments < 1.0 mm in size.When filtering the water, mussels are unable to distinguish the small fragments during feeding.When the concentration of microplastic particles in the water is high, it is naturally taken up by the zebra mussel and mistakenly consumed as plankton.
In terms of colour, five colours were found -black, red, blue, yellow and transparent.In the samples, black and red microplastic particles predominate (Figure 2).As blue microplastics were detected in both control groups, which should be due to microplastic contamination from the environment, the blue microplastics detected in the zebra mussel samples were excluded from the data processing to ensure the accuracy of the results.The percentage distribution of different microplastic colours by zebra mussels' size classes was studied (Figure 3).Zebra mussels were divided into three size classes according to the body length: 15.1-20 mm, 20.1-25 mm and 25.1-30 mm.In the zebra mussels of the smallest size class (15.1-20 mm), about 50% of the accumulated microplastic was black, whereas in the size class 25.1-30 mm red microplastic prevailed.The three groups of zebra mussels did not differ significantly in the proportion of yellow and transparent microplastics.In terms of shape, three types of microplastics were found in the samples: fragments, fibers and pellets.Microplastic fragments are mainly solid particles, flat and irregular in shape (figure 4B).Microplastic fibers are long, thin and twisted in appearance (figure 4C).Plastic pellets originate from plastic particles used in large quantities in the production of plastics (figure 4A).These pre-production plastic particles are sometimes referred to as "nurdles" [27].Figure 5 shows that fragments predominate in the zebra mussel samples, followed by fibers, and pellets.Mussels from the 25.1-30 mm size class had the highest proportion of fragments, while the one medium length (20.1-25 mm) had more fibers and pellets (figure 6).Pastorino et al. [12] demonstrate that the amount of microplastics in zebra mussels sampled close to the wastewater treatment plant were higher compared to the other two sampling sites.The most common microplastics in all the three locations were fibers, while the fragments were more common in samples from highly anthropogenically impacted sites.The sand pit lake Kazichene is highly urbanized because of its proximity to the village of Kazichene and the hydro-park for aquatic sports.The lake is also preferred place for angling.All these activities highly increase the anthropogenic impact on the lake and this is probably the reason why the fragments are the most common type of microplastics.Fiber microplastics mainly come from the degradation of broken fishing nets or ropes.Microplastic fragments mainly originate from the breakage and decomposition of plastic products such as plastic bottles.Numerous pellets are lost during production, transport and storage [28].Spilled or discarded pellets make their way into drainage systems, circulate through the surface waters of rivers, lakes, seas, and oceans [29].Pellet microplastics can also come from domestic wastewater, and products such as toothpaste, shower gel, and facial cleansers which contain high numbers of microplastic particles [30].

Relationship between accumulated microplastics and zebra mussel morphology
The size of the accumulated microplastics varies between 0.25 mm and 5 mm indicating that the zebra mussel can filter microplastics in a wide range of sizes (table 1).
The correlation between zebra mussel meat weight and the amount of accumulated microplastics is shown in figure 7. The correlation coefficient (r = 0.43) indicates a moderate degree of correlation.
Figure 8 shows the relationship between shell length and microplastic accumulation in zebra mussels.The correlation coefficient (r = 0.30) shows that there is a weak correlation between the two parameters.
As no strong relationship between the size (weight) of the mussel and the amount of the accumulated particles was found, the hypothesis that bigger mussels would accumulate higher amount of microplastics was rejected.
There are different studies aiming to prove whether dreissenid mussels could be used as bioindicator to monitor the microplastic pollution in freshwater environment.Pastorino et al. [12] showed that zebra mussels may provide a valid tool to monitor microplastics pollution in lakes.The authors also encourage the use of the invasive species as a bioindicator as a sustainable tool to manage aquatic ecosystems, reducing the use of native species.On the other hand, the initial conclusion of Hoellein et al. [13] was that these mussels do not serve as indicators of microplastic pollution.The reason for this was that the variation in microplastics among individuals, size classes, and time periods was higher or equal to the variation among sites.In our study microplastics were detected in all the samples of zebra mussels from Lake Kazichene, which indicates that this mussel has the potential to be used for monitoring microplastic pollution in the standing freshwater bodies of Bulgaria.To most accurately assess the zebra mussel's potential as a bioindicator of microplastic contamination, further studies are needed on the interaction between individual mussel characteristics such as size, age, filtration, etc. and their interaction with environmental factors such as temperature, season, food availability and quality, etc. 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Conclusion
The average amount of microplastics in zebra mussels from Lake Kazichene was 38.6 pieces per mussel.Three types of shapes were found: pellets, fragments and fibers, with the highest proportion of the fragments, followed by the fibers.The length of the fibers varied from 0.25 to 5 mm.The colours of the observed microplastic particles were black, red, yellow and transparent.The black microplastics were mostly found in the zebra mussels from size class 15.1-20 mm, while the red pieces dominated in size class 25.1-30 mm.No significant relationship was found between zebra mussel meat weight/ shell length and the amount of accumulated microplastics.The importance of this study was to investigate the applicability of D. polymorpha for bioindication of microplastic pollution in Bulgarian freshwater bodies.The adaptation and successful implementation of a methodology for studying the accumulation of microplastics in freshwater mussels lays the foundation for this type of research in freshwater bodies in Bulgaria and future monitoring of microplastic contamination in freshwater environment.As a species with a worldwide distribution, D. polymorpha serves as a suitable subject for future monitoring and comparison of microplastic accumulation in various types of standing water bodies across Europe and globally.

Figure 2 .Figure 3 .
Figure 2. Distribution of microplastics by colour in D. polymorpha from Lake Kazichene

Figure 5 .
Figure 5. Distribution of microplastics by shape in D. polymorpha from Lake Kazichene

Figure 6 .
Figure 6.Percentage distribution of microplastics of different shapes in the different size classes of D. polymorpha from Lake Kazichene

Table 1 .
The length of microplastic fibers from D. polymorpha samples, from the sand pit Lake Kazichene a SDstandard deviation.