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

doi:10.21203/rs.3.rs-1892051/v1

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

https://doi.org/10.21203/rs.3.rs-1892051/v1

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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, which is the largest wetland in North India and is a great home for migratory birds and aquatic animals, and the water of its canal that supplies water to the semiarid part of India's largest state, microplastics in the water were analyzed. Apart from seasonal variation, the fish gut of C. carpio caught from the fishing site of the canal has also been analysed. Between sites 1 and 2, no significant difference was found in the quantity of microplastics, but in season variation, winter showed a higher significant level than summer and the rainy season. The wetland and canal were dominated by two types of plastics: HDPE and nylon. The conformational test, along with FTIR and GC-MS, was also performed. From the gut of C. carpio, microplastics of less than 5mm in size were also recovered. The percentage of recovery was 7% of the total fish analysed. 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.

Emerging pollutant

Harike wetland

Microplastic

Ramsar wetland

C. carpio

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 (Horton & Barnes, 2020). Microplastic is a degraded synthetic polymer that varies in size from 5mm to a few microns (Issac & Kandasubramanian, 2021). 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 (Yuan et al., 2022). 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 well-known, 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 (de Sá et al., 2018). Microplastics may also leach additives, which have been shown to be hazardous, endocrine disrupting, or potential carcinogenic (Campanale et al., 2020). 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 (Naqash et al., 2020).

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 (Prasad et al., 2002). 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 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 (Li et al., 2018; Parker et al., 2021).

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 (Von Moos et al., 2012), (Watts et al., 2014), (Lusher et al., 2013), (Besseling et al., 2013) 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 (Cole et al., 2013), (Hämer et al., 2014), (Setälä et al., 2014). 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 (Van Cauwenberghe & Janssen, 2014). 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 (Hämer et al., 2014). 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 (Watts et al., 2014). Capability of MPs towards bioaccumulation increases with decreasing size (Wagner et al., 2014). 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 (Au et al., 2017). 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 (Hämer et al., 2014). On the other hand, some studies have shown untoward effects of microplastic particles on juvenile and adult fish (Rochman et al., 2013), (de Sá et al., 2015).

Besides the problem of their accumulation in organisms, there is yet another serious issue related to microplastics. Plastics contain a multitude of chemical additives (Dekiff et al., 2014) and adsorb organic contaminants from the surrounding media (Bakir et al., 2012). Since these compounds can transfer to organisms upon ingestion, microplastics act as vectors for other organic pollutants (Zarfl & Matthies, 2010) and are, therefore, a source of wildlife exposure to these chemicals (Teuten et al., 2009).

The identification and measurement of microplastics in the environment begins with the collection of representative samples. Depending on the sample type, several sampling procedures might be utilized. Harike wetland being one of the largest wetlands in India forms an area of about 4100ha (Bhardwaj et al., 2017). The site was selected in such a way that what effect does it have on the wetland and the canal coming out of it, so that not only the wetland but also the way of its services can be analyzed. Each location was coordinated and illustrated as shown in Fig. 1. Forest department monitoring station which is located near Gurdwara Nanaksar Sahib was selected as sampling site one. Sampling was done by guard boat with the permission of Forest department Chandigarh. Harike canal selected as second sampling site because it is an authorized site for fishing, supplying water to Rajasthan for irrigation and drinking.

Due to the low concentration of MPs in the water, volume reduced sample method followed in which volume-reduced samples are usually obtained by filtering large volumes of water with plankton net with mesh size 60 µm. The net was tow with the boat for about 5×10^− 1Km² from the shore and water was filtered up to 15 cm depth from surface to collect floating debris. After towing, the net was properly packed in such a way that there would be no secondary contamination. Net was washed in a closed chamber so that even the fine particles adhere to it could be collected in the bath tub. 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 of the steps were carried out under a fume hood, and synthetic-free clothing and gloves were worn at all times and all glassware wash regularly with double distilled water.

Under a binocular stereoscope, the samples were visually evaluated for the presence of microplastic. Plastic particles were extensively examined in the samples, including the edge of the Petri dish, which is where micro-plastic particles generally cling. The particles were photographed and counted. All macro particles are designated from an alphabet then placed over slide and observed under binocular stereo microscope (radicle, RSM-9) to get size and shape (software – Micaps) (Fig. 2, a). After getting size point hot needle checked whether the material is synthetic or not, if the material is synthetic, it will shrink and reduce size (Fig. 2, b₁ & b₂) (Nuelle et al., 2014).

Filtered residue underwent an oxidation process to breakdown labile organic matter and a density separation process to separate lighter plastic particles from denser, mostly inorganic components (Rodrigues et al., 2018).

Oxidation was performed by using 4ml of 30% of H₂O₂ and density based gradient was created by adding NaCl solution with density 1.8 g/cm³, so that possible microplastic particles could easily float on top (Hosseini et al., 2020). The top layer of the sample after density gradient process was subjected to centrifugation at 3500rpm for 15min. Then the supernatant collected by assuming the plastic particle to be in it due to low density and dried in a dryer at 55^oC for 20min. The dried sample was weighed and equally divided for spectrometry analysis that is ATR-FTR and Pyr-CG/MS in order to get confirmation about presence and type of plastic in sample. This process repeated for all three-season monsoon, summer and winter season from March 2019 to Feb 2020.

Traces of plastic particle in gut content of C. carpio

To analyze occurrence and amount of microplastic ingested by fish, a total 171 individual (from March 2019 to Jan 2020) C. carpio aged 1 + were collected from fishing sites of canal where wetland water is flowing towards Rajasthan state for agricultural and domestic uses. Fish gut collected on the site by maintaining precaution to avoid secondary contamination packed in ice box to carry for the lab. In lab each gut externally washed with distilled water and dissect to collect gut content. Under a binocular stereoscope (radicle, RSM-9, software - Micaps), the samples were visually evaluated for the presence of microplastic, with distilled water used to rinse the GIT contents and aid in the identification of plastic particles.

The aim of in situ sampling was to assess microplastic debris in wetland and their penetration in biological system. Water filtration of two different sites is done by towing for three seasons which are - Summer (May 2018), Monsoon (August 2018) and Winter (January 2019). Summer season was hot and dry whereas winds in rainy season with precipitation damp climate recorded. Weather was calm in winter and temperatures usually hovering between 0°C and 16°C. Highest flow was observed in rainy season followed by winter. To maximize the water surface area analyzed and limit forces induced by strong currents, the net was only immersed halfway. Only the floating debris superior to 6 cm is collected and the debris, including macro-plastics, that sink or settle on the riverbed are neglected.

The banks of the wetland were covered with water hyacinth, which has become a common occurrence for any fresh water body. Due to the small mesh size of the net, many large particles were collected in every season, out of which many were green leaves and other organic waste. It was difficult to select synthetic polymers as there were clear and natural-colored particles.

Microplastics

The initial study was completed along with a morphological examination. This allowed 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 exhaustive morphological examination of particles, in summer season a total 7 particles from site 1 (Gurudwara Nanaksar sahib, Harike wetland) and 9 from site 2 (Indira Gandhi canal/ Rajasthan canal) were grab for microscopic observation. These particles were illuminated, lightweight fibers, films and irregular in shape varied from 1 mm to 4 mm in size. The most frequent particles morphologically observed were films followed by fragments and fibers. These particles passed through hot needle test under observation of binocular stereo microscope and only 6 particles of site 1 and 5 from site 2 shrunk and confirm as synthetic material because as synthetic things shrink when coming in contact with hot needle (De Witte et al., 2014). A total 9+6 from site 1 and 2 in monsoon and 15+11 in winter season particles sorted out respectively by same procedure. This time all particles were confirmed as synthetic by point hot needle test. In rainy season out of which 2 particles (K and N, Fig.2-a₂) were larger in size, 8.85mm and 9.85mm so not consider as microplastic. Rest of the particles carefully preserved in glass container for spectroscopic analysis.

In rainy season 8.11mg and in winter 6.45mg wt. of particles sized more than 305 µm recovered after sieving. Same 7.9 and 9.12mg for size between 305 to 200 µm, 4.15 and 6.88 for 100 to 200 µm and 4.2 and 6.97mg below 100 but larger than 50 µm was estimated for rainy and winter season from site one and two respectively. A comparative table for both sites and all seasons is given below (Table 1) These particles divided in two equal quantities for vibrational and mass spectroscopy. Paired sample t-test was to be applied for two sites and it was found that the significant value is greater than 0.05 (p > 0.05) so there is no quantitative significant difference between the two sites. The second analysis took place between seasons and Statistical difference was found in 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 microplastic quantity. Winter being near to post rainy season therefore high contaminants was present in 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.

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.

Mesh Size (µm)	Site 1			Site 2
Mesh Size (µm)	Summer	Rainy	Winter	Summer	Rainy	Winter
>305	5.6	8.11	6.45	5.11	6.25	5.2
>200	7.2	7.9	9.12	6.9	7.04	6.4
>100	5.56	4.42	6.88	3.29	9.1	5.87
>60	3.56	4.2	6.97	3.52	8.8	6.72

Vibrational spectroscopy (ATR-FTIR)

To set up a general spectral database, 15 different common polymer beads samples from plastic industry were measured via attenuated total reflection (ATR)-FTIR spectroscopy. When spectra of these beads compared to the spectra of scientifically sourced polymers, the appearance and number of identifiable wavenumbers were nearly identical. After that MPs were detected from all effort i.e., morphological examination and sieved from different mesh size samples measured via ATR-FTIR. Table 3 summarizes the main ATR-FTIR absorption
bands of the samples analyzed in this study. All visible particles of different seasons were shows various characteristic bands matching with high density polyethylene (HDPE) and polyamide (Nylon-6). The typical HDPE peaks were 719 cm^-1δ_rocking^*C - H (−CH₂−), 2854 cm^-1, 2925 cm^-1 ν_as^** and ν_s^*** −CH₂− and a peak 1472cm^-1 δ_{s in-plane} C−H. Overlapping (ORIGINPro9 64-bit) these peaks with the spectra of standard plastic beads (HDPE) resulted in approximate matching (Figure 3 - b). Hence hot needle test gave confirmation about the synthetic nature of particles whereas ATR-FTIR gave an indication about the functional groups of HDPE. Low-density polyethylene (LDPE) and high-density polyethylene (HDPE), both materials only showed small differences by additional spectra 1377 cm^-1 in LDPE.

In all size sieved residue second type of polymer polyamide (Nylon-6) identified with reference spectra 675cm^-1 N-H bend, 1196cm^-1 CH₂ bending, 1263cm^-1 N-H bend, C-N stretch, 1369cm^-1 νs CH₂ bending, 1465cm^-1 νs* CH₂, 1538cm^-1 δin plane -NH and ν C-N, 1636 cm^-1 ν C=O stretch, 2855cm^-1 νs, 2932 cm^-1 νas and 3298 cm^-1N-H stretch.

*Scissoring

**stretching asymmetrical

*** stretching symmetrical

Except these spectra some other spectra were also observed such as 1741cm^-1, 1840cm^-1, 1892cm^-1, 1959cm^-1 which are for monosubstituted aromatic rings found in polystyrene (PS) but further it could not be confirmed by Pyr. GC-MS.

GC-MS analysis

Single visible particles each from all suspected plastic category by ATR-FTIR and parts of particle size above 305 µm and between 100 to 200 µm sieved residue analyzed by GC-MS. As the degradation pattern of polymer remains identical, identification of compounds was carried out by studying and comparison of mass spectra of sample with the available fragmentation pattern of polymers in WILEY and NIST library out of which the most matching one. The fragmentation of HDPE includes 1-heptene, 1,9-decadiene, 1-Decene, n-decane, 1-undecene, 1-tetradecene, 1-eicosene, 1-triacontene, 1-tetracontene, 1-hentetracontene. Mostly polyamide breaks in 1- pentenenitrile, pentanenitrile, 6-aminohexanenitrile, €-caprolactam, N-(5-cyanopentyl) butyramide, N-(5-cyanopentyl) hex-5-enamide, N-(5-cyanopentyl)hexanamide, 6-acetamido-N (5cyanopentyl) hexanamide, 6-butyramido-N-(5-cyano pentyl) hexanamide, N-(5- cyanopentyl)-6- hexanamide) fragments.

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 1-heptene, peak no. 83, 69, 55, 41 and 27 which usually refers 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 tetradecane 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 particles morphologically observed were films followed by fragments and fibers. These particles are major component of microplastics delivered by atmospheric deposition (Dris et al., 2016). In addition to atmospheric deposition, riverine (Baldwin et al., 2016) (wastewater/washer effluents in rivers) (Browne et al., 2011), shoreline runoff, litter and uses of cement bags at bank of wetland to avoid erosion are speculated sources of debris to surface water sites.

The findings of this study show that both ATR-FTIR and GCMS are capable of characterizing the chemical composition of ambient MPs and can 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, time-consuming literature study or professional experience may be necessary in some circumstances.

Our results reveal the presence of microplastics in water throughout wetland, and variation of microplastic number between seasons and sampling sites. In the case of macroplastic, it is usually assumed that approximately 80% of trash originates from land-based sources (Allsopp et al., 2006).

In carp

For the separation of gut contents, guts were measured having mean weight of 5.50gm and each fish gut was carefully washed with distilled water followed by
incision and content was collected. Visible particles with size 1.17mm, 1.81mm, 2.91mm, 3.38mm, 2.60mm, 4.72mm, 2.51mm, 1.13mm were separated during physical sorting
and kept separately for further analysis. 11 particles which were separated initially with the help of foreceps from 7 fish gut on an average 1.57 particles/gut, they were
subjected to hot needle test for confirmation of plastic nature where they found same. Point 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 microplastic in them out of 171 fish, so 4.09% of fish were carrying visible microplastic.

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 (Jovanović et al., 2018). Therefore, such particles might be ingested accidently by fish or misinterpreted as prey or even transferred through other 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 (de Vries et al., 2020). 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 (Koongolla et al., 2020).

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 microplastics. The impact of industry and mismanagement is not only on the local environment but also on the protected area and wildlife at a faraway place. To understand this, Harike Wetland was one of the most appropriate and large wetland. The study of the harike wetland and its canal, has highlighted macroplastic and microplastic contamination. Presence of microplastics (fragments and anthropogenic fibers) in surface waters is related to the presence of several textile and plastics industries and illigal deposition of waste in every cities near rivers that sustain wetlands - Beas and Satluj. The sorting of plastic debris highlighted that plastic bags, nylon fibers and small pouch of different products such as shampoo schache blades spices tobaccos and HDPE made products were dominant. Plastic waste mismanagement is a critical issue which has important environmental consequences. Wetland protection and restoration agencies will have to start working on its mitigation and control, as well as see how it is having an impact on wetland services.

This study, combining both microplastic assessments in wetlands and in its traces in 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 microplastic contamination, it has been done on a Ramsar wetland and is the first of its kind study of continuous monitoring which shows the infiltration of microplastics in the protected area. Since we should not hesitate to accept that plastic production will increase in the next few years and the degradation of plastic is happening continuously in the environment, due to which the load of microplastics is going to increase and it has reached our health in toxic form or is just about to reach.

Authors Contributions: Shaista Manzoor- Experiment, data analysis, Rahul Singh- conceptualization, methodology and writing- original draft

Ethical Approval: Not Applicable

Consent to participate: Not Applicable

Consent to Publish: Not Applicable

Funding: No any funding received from any agency for this work.

Competing interest: The authors declare no competing interests.

Data availability: Not applicable.

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

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