Spatiotemporal microplastic occurrence study of Harike wetland, A Ramsar wetland of India

: Ramsar wetlands are one of the least investigated areas for microplastic contamination. Harike wetland is located downstream of the confluence of the Beas and Sutlej rivers. Rivers are nature's drainage systems, which collect waste from everywhere and move ahead. On the same note, the Harike wetland, the largest wetland in North India, is an ideal home for migratory birds and aquatic animals. The water in the canal and the fish living within it have been examined for microplastics. Results revealed that the number of microplastics did not significantly differ between sites 1 and 2, but showed a seasonal variation with higher levels observed in the winter season compared to summer and the rainy season. The most common types of plastics detected in the wetland and canal were HDPE and nylon, which were further analyzed using conformational tests, ATR-FTIR, and GC-MS techniques. Additionally, microplastics measuring less than 5mm in size were recovered from the gut of Cyprinus carpio fish, with a 7% recovery rate from the total number of fish analyzed. Although this percentage may seem low, it cannot be ignored given the potential impact on the aquatic environment. This percentage may be less, but it is not ignorable. Indeed, the amount of poorly managed plastic garbage generated by people living around or illegal outskirts garbage dumping near rivers and tributaries is a potential source of aquatic debris.


Introduction
Microplastic (MP) particles are emerging pollutants in the environment, and analyzing them is a new subject of area.Since the first investigation in the 1970s in the marine ecosystem, microplastics are becoming increasingly investigated globally [1].Microplastic is a degraded synthetic polymer that varies in size from 5mm to a few microns [2].Many studies revealed that microplastic are present from beach sediments to deep sea sediment and some studies have also registered its strong appearance in fresh water [3].With more information about the accumulation of microplastics in aquatic ecosystems, people are becoming more aware of the environmental consequences.The absorption of these extremely small particles by a wide range of marine creatures is wellknown, demanding risk assessment approach.It is critical to have accurate data on the presence of microplastics in marine and freshwater ecosystems.There are currently no harmonized and defined techniques in place for sampling, analysis, evaluation, and quantification of microplastics in aquatic samples.MP's small size allows it to be consumed by a various species with different feeding habits and techniques, resulting in direct physical injury and probable toxicity [4].Microplastics may also leach additives, which have been shown to be hazardous, endocrine disrupting, or potential carcinogenic [5].MPs have the ability to enrich persistent organic pollutants (POPs) and possibly hazardous components by adsorbing them at higher quantities than those found in the surrounding environment [6].
Analysis of microplastics in wetlands would be an ideal representation to determine the level of contamination in fresh water bodies and the organisms living in them.The journey of microplastics, like other contaminants, often originates from the land and ends up in the aquatic body.Wetlands are important interface between land and water and are essential to maintain ecological processes, life support systems and bio-resource production.Wetlands are considered to have unique ecological features which provide numerous products and services to humanity [7].Ecosystem goods provided by the wetlands mainly include: water for irrigation; fisheries; non-timber forest products; water supply; and recreation.Major services include: carbon sequestration, flood control, groundwater recharge, nutrient removal, toxics retention and biodiversity maintenance.While there are a huge number of studies on microplastics pollution in the marine environment, there has been a dearth of information on , 01048 (2024) BIO Web of Conferences https://doi.org/10.1051/bioconf/2024860104886 RTBS-2023 microplastics in freshwater systems.In the light of the rapid recent developments in microplastic research, various studies to improve understanding on microplastic quantities and distribution in freshwater and treated water sources have been initiated by many workers.Various workers have also recently carried out several studies on freshwater, terrestrial and human health aspects [8], [9].Microplastics play a more consequential and important ecological part to play in the aquatic food-web.The small size of microplastics facilitates easy uptake by organisms compared to larger ones.Microplastic ingestion has been reported for many different species, such as mussels, lugworms, crabs, seabirds, and fish [10], [11], [12], [13].As smaller organisms, such as zooplanktons, isopoda, or mysid shrimps, which all are at the bottom of the food chain can easily consume these smaller particles, bio-magnification of microplastics is expected [14], [15], [16].As such, one of the major concerns among the scientific community regarding the presence of microplastics in various ecosystems is their bioaccumulation, in particular bio-magnification and trophic transference.Microplastics have been detected in mussels, which are cultured for human consumption.Due to smaller size, microplastics are less likely to block the gastro-intestinal system, and even small crustaceans such as isopods can excrete digested microplastics [15].However, retention time of microplastics in an organism depends on the uptake route as well as the type of the organism and if the retention time is long, the ingested microplastics can be transferred to the next trophic level [11].Capability of MPs towards bioaccumulation increases with decreasing size [17].Reduced size of microplastics makes them more susceptible to ingestion by diverse group of organisms ranging from planktons, fish, birds and even mammals, accumulating throughout the food web [18].In some organisms microplastics do not seem to affect mortality of organisms such as isopods.This may be due to the ability of these organisms to excrete microplastics [15].On the other hand, some studies have shown untoward effects of microplastic particles on juvenile and adult fish [19], [20].
Besides the problem of their accumulation in organisms, there is yet another serious issue related to microplastics.Plastics contain a multitude of chemical additives [21] and adsorb organic contaminants from the surrounding media [22].Since these compounds can transfer to organisms upon ingestion, microplastics act as vectors for other organic pollutants [23] and are, therefore, a source of wildlife exposure to these chemicals [24].The aim of this research article is to investigate the seasonal variation in the presence, distribution, and abundance of microplastics in the Harike wetland.Apart from seasonal variation, the fish gut of Cyprinus carpio caught from the fishing site of the canal has also been analyzed.The study seeks to identify the sources of microplastic pollution in the wetland and to evaluate the potential ecological impacts of this pollution on the wetland ecosystem.

Methodology
The identification and measurement of MPs in the environment begin with the collection of representative samples.Depending on the sample type, several sampling procedures might be utilized.Harike wetland, one of India's most significant wetlands, forms an area of about 4100ha.This wetland came into being when headwork was being built across the rivers Beas and Sutlej in the year 1952.The aim of building the headwork was storing water, drinking water supply and irrigation purpose to the Rajasthan state and south Punjab [25].To analyze the wetland and the services provided, the wetland and canal were considered when choosing the location.Each location was coordinated and illustrated, as shown in Figure 1.The forest department monitoring station near Gurudwara Nanaksar Sahib (31°08′29.6'N74°57′05.1'E)was selected as the first sampling site.Sampling was done by guard boat with the permission of the Forest department Chandigarh.Harike canal (31°07′50.3'N74°56′59.8'E)was selected as the second sampling site because it is an authorized site for fishing, supplying water to Rajasthan for irrigation and drinking.The rationale for choosing two sampling locations is to offer a point of reference for the protected portion of the wetland in one location (Gurudwara), while the other location (Harike canal) was included due to the importance of the water for both drinking and irrigation purposes.Due to the low concentration of MPs in the water, volume reduced sample method followed in which volumereduced samples are usually obtained by filtering large volumes of water with plankton net (S.K Appliances) mesh size 60 µm.The net was towed with a boat up to 5×10 -1 km 2 from the shore.The water was filtered to a depth of 15 cm from the surface to collect the floating debris.After towing, the net was adequately packed so there would be no secondary contamination.The net was washed in a closed chamber, and fine particles that adhered to it were collected in the bathtub.Both organic and suspended synthetic polymers were present in the bathtub, so the particles whose size was visible were carefully separated by forceps.The rest of the water was sieved by different mesh sizes like 305µm, 200µm, and 100µm.All the steps were carried out under a fume hood, synthetic-free clothing and gloves were worn at all times, and all glassware was washed regularly with double distilled water.Under a binocular stereoscope (radicle, RSM-9), the samples were visually evaluated for the presence of MP.Plastic particles were extensively examined in the samples, including the Petri dish's edge, where micro-plastic particles generally cling.The particles were photographed and counted.All macro particles are designated from an alphabet, then placed over the slide and observed under a binocular stereo microscope to get size and shape (software -Micaps) (Figure 2 (A)).Once the samples have been cleaned of any organic matter, the next step is to verify that the particles are indeed plastic.One way to do this is by using a hot needle test.A small portion of the sample is placed on a heat-resistant surface, and a hot needle is applied to the particle.If the particle melts or deforms, it is likely made of plastic.(Figure 2 (B1, B2).Filtered residue underwent an oxidation process to break down labile organic matter and a density separation process to separate lighter plastic particles from denser, mostly inorganic components [27].
Oxidation was performed by using 4ml of 30% of H2O2.The density-based gradient was created by adding NaCl solution with a density of 1.8 g/cm 3 , so that possible MP particles could easily float on top [28].After the density gradient process, the top layer of the sample was subjected to centrifugation at 3500rpm for 15min.The supernatant was collected by assuming the plastic particle was low density and dried in a dryer at 55°C for 20min.
The dried sample was weighed and equally divided for spectrometry analysis, ATR-FTIR and Pyr-GC/MS, to confirm the presence and type of plastic in the sample.ATR-FTIR spectroscopy can be used to identify the chemical composition of microplastics by comparing the measured spectra to a reference library of spectra for different types of plastics. of microplastics.It involves heating the sample to a high temperature in the absence of oxygen, which causes the plastic to break down into its constituent components.The resulting gases are then analyzed by gas chromatography and mass spectrometry to identify the chemical composition of the plastic.This process was repeated for all three-season monsoon, summer, and winter seasons.

MPs' collection from fish gut
To analyze the occurrence and amount of MP ingested by fish, a total of 171 individuals.Cyprinus carpio aged 1+ were collected from fishing sites of the canal where wetland water flows towards Rajasthan state for agricultural and domestic uses.Fish gut was collected on the site by maintaining precautions to avoid secondary contamination and packed in an ice box for the lab.Each gut was externally washed with distilled water and dissected in the lab to collect gut content.The samples were observed for MP particles under a binocular stereoscope (radicle, RSM-9, software -Micaps).Plastic particles were extensively examined in the samples, including the Petri dish's edge, where micro-plastic particles generally cling.The particles were photographed and counted.All macro particles are designated from an alphabet.Rest of the material, a digestion protocol was adapted from the procedure given by a previous study to increase the efficiency of plastic extraction [29].

Results and Discussion
In situ sampling assesses MP debris in wetlands and its penetration into the biological system.Water filtration of two different sites is done by towing for three seasons which are -Summer, Monsoon and Winter.The summer season was hot and dry, whereas winds in the rainy season with precipitation damp climate recorded.Weather was calm in winter, and temperatures usually hovered between 0°C and 16°C.The highest flow was observed in the rainy season, followed by winter.The net was only immersed halfway to maximize the water surface area analyzed and limit forces induced by strong currents.Only the floating debris superior to 6 cm is collected, and the debris, including MPs, that sinks or settles on the riverbed is neglected.The wetland banks were covered with water hyacinth, which has become common for any freshwater body.Due to the small mesh size of the net, many large particles were collected every season, out of which many were green leaves and other organic waste.It was challenging to select synthetic polymers as there were clear and natural-colored particles.The initial study was completed along with a morphological examination which allowed the material to be assessed, such as whether it was a synthetic or natural organic polymer.These particles were recognized alphabetically for future records (figure 2, a).
In the lab, after an exhaustive morphological examination of particles, in the summer season, a total of seven particles from site 1 (Harike wetland) and nine from site 2 (Gurudwara) were grabbed for microscopic observation.These particles were illuminated, lightweight fibres, films and irregular shapes varied from 1 mm to 4 mm in size.The most frequent particle morphologically observed were films, fragments, and fibres.These particles passed through the hot needle test under the observation of a binocular stereo microscope, and only six particles of site 1 and 5 from site 2 shrunk and confirm as synthetic material because synthetic things shrink when coming in contact with the hot needle [26].The same procedure sorted out a total of 9+6 from sites 1 and 2 in monsoon and 15+11 in winter season particles.This time all particles were confirmed as synthetic by the point hot needle test.In the rainy season, two particles (K and N) were more prominent in size, 8.85 mm and 9.85 mm, so they were not considered MP.The remaining particles were carefully preserved in a glass container for spectroscopic analysis.Accidentally from Harike wetland, sieved particles missed to analyze in the summer season.In the rainy season, 8.11mg, and in winter, 6.45 mg wt. of particles sized more than 305 µm recovered after sieving.Same 7.9 and 9.12 mg for sizes between 305 to 200 µm, 4.15 and 6.88 for 100 to 200 µm and 4.2 and 6.97 mg below 100 but larger than 50 µm was estimated for rainy and winter seasons, respectively.A comparative table for both sites and all seasons is given below (Table 1).These particles are divided into two equal quantities for vibrational and mass spectroscopy.A paired sample t-test was to be applied to two sites, and it found that the significant value is greater than 0.05 (p > 0.05), so there is no significant quantitative difference between the two sites.The second analysis took place between seasons, and the statistical difference was found in the rainy and winter season for site 2 (Harike canal) as P<0.05, but there was no difference for site 1 (Near Gurudwara Nanaksar sahib) with P>0.05 for MP quantity.Winter is near the post-rainy season; therefore, high contaminants were present in the water.It appears that the contaminants brought by the rivers are not only affecting the Harike wetlands but also reaching other states which are miles away through the canal.Statistical difference was found in rainy and winter season as P<0.05 for microplastic quantity.Out of sampling one rainy season (S1R) and sampling two rainy season (S2R), S2R is greater for microplastic quantity due to it being the ending of the rainy season.There are significantly less contaminants in pre-rainy season but after rainfall the washoff brings more contaminants along from different parts including mountains, hills, aquatic bodies including small streams, tributaries.Post rainy season, the level of contaminants in water increase as whole washoff gets collected thereby increasing the chance of microplastic with varying types in water.Turbidity being another factor in addition to flow of water, it also contributes due to the factor that during rainy season the turbidity of wetland is more leading to more contaminants in water.Following in winter season, the sampling one winter season (S1W) is near to ending of rainy season which means there will be almost similar contaminant level with a slope of it gradual decreasing.

Vibrational spectroscopy (ATR-FTIR)
To set up a general spectral database, 15 different standard polymers (PET: polyethylene terepthalate, PS: polystyrene, HDPE: high density polyethylene, LDPE: low density polyethylene, LLDPE: linear low density, PP: polypropylene, HIPS: high impact polystyrene, CA: cellulose acetate, EVA: ethylene vinyl acetate, ABS: acrylonitrile butadiene styrene, PU: polyurethane, PVC-f: poly vinyl chloride flexible, PVC-r: poly vinyl chloride rigid, PA6: polyamide 6, PA6.6: polyamide 6.6) beads samples from the plastic industry were measured via attenuated total reflection (ATR)-FTIR spectroscopy.When the spectra of these beads were compared to the spectra of scientifically sourced polymers, the appearance and number of identifiable wavenumbers were nearly identical (data not shown).After that, MPs were detected, i.e., morphological examination and sieved from different mesh size samples measured via ATR-FTIR [30].All visible particles of different seasons showed various characteristic bands matching with high-density polyethylene (HDPE) and polyamide (Nylon-6).The typical HDPE peaks were 723.33cm -1 δrocking* C -H (−CH2−), 2854.74cm - , 2925.15cm -1 νas** and νs*** −CH2− and two peaks between δas and δs in-plane C−H 1300-1400 cm -1 .Hence hot needle test confirmed the synthetic nature of particles, whereas ATR-FTIR indicated the functional groups of HDPE.Low-density polyethylene (LDPE) and high-density polyethylene (HDPE), both showed minor differences between the spectra.All the peaks mentioned along with the functional groups representing the particles as HDPE and LDPE are same.However, the presence of a characteristic peak at 1377 cm -1 confirmed it as LDPE.
In addition to typical HDPE spectra in all size sieved residue second type of polymer polyamide (Nylon-6) identified with reference spectra 1573.97cmExcept for these spectra, some other spectra were also observed, such as 1741 cm -1 , 1840 cm -1 , 1892 cm -1 , and 1959 cm -1 , which are for mono-substituted aromatic rings found in polystyrene (PS), but further Pyr GC-MS could not confirm it.
The first fragments matched in the mass spectrum of the sample were 1-heptene, a very common degraded fragment of HDPE.An intensity of 98 m/z was low in the graph, but this is due to degradation when the bonds break and the alkanes separate.In the analysis of the expanded mass spectrum of peak no.83, 69, 55, 41 and 27 usually refer to the shortened mass due to the separation of methane, ethane propane and so on.Another HDPE product 1-decene observed at 8.6 min with characteristic fragment ion m/z 140.Similarly, other fragments of HDPE, such as tetra-decane m/z 198 with its degraded fragment peaks 155, 127, 99, 85, 71, 57, 43 and undecene m/z 168 with its fragments m/z 140, 111, 69,43.
The most frequent particle morphologically observed were films, fragments, and fibres.These particles are a significant component of MPs delivered by atmospheric deposition [31].In addition to atmospheric deposition, , 01048 (2024) BIO Web of Conferences https://doi.org/10.1051/bioconf/2024860104886 RTBS-2023 riverine [32] (wastewater/washer effluents in rivers) [33], shoreline runoff, litter and use of cement bags at the bank of wetlands to avoid erosion are speculated sources of debris to surface water sites.The findings of this study show that both ATR-FTIR and GCMS can characterize the chemical composition of ambient MPs and complement one another.Organic and inorganic contaminants in IR spectra might overlap polymer bands and obstruct spectroscopic evaluation, which is a drawback of FTIR.Because GCMS polymer databases are currently being built and are not as well-known as FTIR polymer databases, a time-consuming literature study or professional experience may be necessary for some circumstances.Our results reveal the presence of MPs in water throughout wetland.Variation of the MP number between seasons and sampling sites has also been discussed.The significant component of MPs is delivered by atmospheric deposition 50.In addition to atmospheric deposition, riverine 26 (wastewater/washer effluents in rivers 51), shoreline runoff, litter and use of cement bags at the bank of wetlands to avoid erosion are speculated sources of debris to surface water sites.In the case of macroplastic, it is usually assumed that approximately 80% of trash originates from land-based sources [34].

In carp
For the separation of gut contents, guts were measured having a mean weight of 5.50gm, and each fish gut was carefully washed with distilled water, followed by Incision and content were collected.Visible particles with size range 1.17mm -4.72mm, were separated during physical sorting.11 particles, separated initially with the help of forceps from 7 fish guts on an average of 1.57 particles/gut, were subjected to a hot needle test to confirm the plastic nature where they found the same.Hot needle test followed by ATR -FTIR characterization, which gave information about the type of polymer in these plastic particles.Out of them, one particle was confirmed as Polystyrene, 3 as polyamide and rest seven particles were confirmed as Polyethylene.Since 7 fish guts showed the presence of MPs in them out of 171 fish, 4.09% of fish were carrying visible MP.Intentional or accidental ingestion of environmental microplastics or ingestion by direct consumption of contaminated prey at lower trophic levels, is responsible for microplastic presence in the fish gut [35].Therefore, such particles might be ingested accidently by fish or misinterpreted as prey or even transferred through other , 01048 (2024) BIO Web of Conferences https://doi.org/10.1051/bioconf/2024860104886 RTBS-2023 animals at lower trophic levels.Smaller the size of microplastic particles, greater is the possibility of accidental ingestion, similarly the quantity of the effect of plastic ingestion relies upon on the particle size and the organism consuming the particle [36].Though many studies have revealed the presence of microplastic in fish gut, however it has to be confirmed whether the particles remain in gut for longer period or they are capable to pass through the gut to non-digestive tissues as well to authenticate its biological impacts [37].

Future perspectives
MP pollution is a serious problem that can harm the environment and potentially impact human health.MP particles can ingested by marine life and other animals which can lead to physical damage to the digestive tract, blockages, and malnutrition [38].In humans, ingestion of MP particles could potentially lead to health problems such as gastrointestinal inflammation.MP particles can adsorb chemicals such as PCBs, DDT, Heavy metals and PAHs from the environment [39].When ingested by living organisms, these chemicals can be released, potentially causing health problems such as endocrine disruption, cancer, and developmental issues.Overtime, microplastic particles can bioaccumulate in the tissues of living organisms, causing toxic effects.Overall, the negative impacts of microplastic particles on human and other life entities are numerous and complex, and it is important to continue researching and addressing this issue to minimize the negative impact on our environment and ecosystems [40].One of the most creative points of this research concerning MP contamination of Harike wetlands and the biota within is the development of innovative monitoring and assessment techniques.For example, scientists are using microplastic tracers to determine the sources and pathways of microplastics in aquatic ecosystems, and they are also using genetic techniques to assess the impacts of MPs on fish populations and their habitats.Another important area of research is the development of strategies for mitigating and preventing MP contamination in wetlands.These strategies can include the reduction of plastic waste, the implementation of wastewater treatment technologies, and the restoration of natural wetland habitats.Overall, a multi-disciplinary approach that involves scientists, policymakers, conservation organizations, and the general public is needed to protect wetlands from microplastic contamination.By combining innovative research with effective conservation strategies, we can work towards a healthier and more sustainable future for our wetland ecosystems.
There are several potential solutions that could help reduce or eliminate microplastic particles in the environment.One of the most effective ways to reduce microplastic pollution is to reduce the use of plastic products in our daily lives [41].This includes using reusable bags, containers, and water bottles, avoiding single-use plastic items, and recycling plastics properly.Wastewater treatment plants can be modified to include filters that capture microplastics before they are discharged into the environment.This can help prevent microplastic particles from entering rivers, lakes, and oceans.Textile manufacturing processes can be modified to reduce the release of microplastic fibers from synthetic fabrics.This can be done by using different materials, changing the manufacturing process, or developing new technologies that capture microfibers.Research is underway to develop biodegradable plastics that can break down naturally and not accumulate in the environment.These materials could potentially replace traditional plastics in certain applications.Scientists are also exploring various techniques for removing existing microplastic particles from the environment, such as using filters or biodegradable materials that capture microplastics.It's important to note that these solutions are not mutually exclusive and that a combination of these approaches may be necessary to effectively address the problem of microplastic pollution.Additionally, it's important for individuals, organizations, and governments to work together to raise awareness of the issue and take action to reduce plastic use and prevent microplastic pollution.

Conclusion
The aim of this was that even in conservative areas where human activity is heavily regulated, there may or may not be a presence of MPs.The impact of industry and mismanagement is not only on the local environment but also on the protected area and wildlife in a faraway place.To understand this, Harike Wetland was one of the most appropriate and extensive wetlands.The study of the Harike wetland and its canal has highlighted macroplastic and MP contamination.The presence of MPs (fragments and anthropogenic fibers) in surface waters is related to the presence of several textile and plastics industries and illegal deposition of waste in every city near rivers that sustain wetlands -Beas and Sutlej.The sorting of plastic debris highlighted that plastic bags, nylon fibres and a small pouch of different products such as shampoo, blades, spices, tobaccos and HDPE-made products were dominant.Plastic waste mismanagement is a critical issue which has significant environmental consequences.Wetland protection and restoration agencies will have to start working on its mitigation and control, as well as see how it impacts wetland services.This study, combining MP assessments in wetlands and their traces in a biological system, has evidenced the close relationship between these two.We, therefore, recommend that the link between these two should not be overlooked in further studies.Although this study is not the first study of MP contamination, it has been done on a Ramsar wetland.It is the first of its kind study of continuous monitoring, which shows the infiltration of MPs in the protected area.Since the production of plastic will rise in the coming years and plastic degradation is occurring continuously in the environment, the MP load will increase and either have already reached or soon will reach our health in a toxic form.

Fig. 1
Fig.1 Map depicting Harike Wetland and sites selected for surface water sampling

Fig. 3
Fig. 3 FT-IR spectra of samples were red curves are depicting spectra of standard polymers and black curves are FTIR spectra of MPs samples

Fig. 2
Fig.2 Chart representing mean of heavy metal concentrations at different sites in comparison with the permissible limits by WHO.

Table . 1
Comparative table for both sites and all seasons for depicting the weight of sample residues (mg) obtained from different mesh size filter cloth -1δin plane -NH and ν C-N, 1639.19 cm -1 ν C=O stretch, 2855.41cm -