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The Gammarus genus, remains of high significance functionally and structurally in the freshwater ecosystems specifically of Northern Hemispheres. It is greatly affected by micro-pollutants. The study has underlined how micropollutant impact upon the ecosystem health using the amphipod species of Gammarus pulex and its behavior studies as a potent bioindicator. In the first sections of the research an introduction to the topic of research has been provided, followed by an in-depth literature review considering the recent academic works conducted within the last 10 years. A secondary methodology of data collection has been considered most suiting the research, as the research topic requires a wide range of knowledge gained from literary sources of repute, and from acclaimed journals for attaining all the objectives. A PRISMA Flowchart has been provided to throw light upon the literature searches made and the reasons behind why some research has been considered in the present study while others have been ignored (Kelly et al., 2016). Finally, data analysis and result interpretation has been conducted to be followed by future research plan and a conclusion of all the important facts obtained in the study.
The Gammarus genus, remains of high significance functionally and structurally in the freshwater ecosystems specifically of Northern Hemispheres (Gerhardt et al., 2011). The presence of the amphipod genus is highly crucial as within the stream detritus processing, a significant rate-limiting stage consists of macro-invertebrate feeding. Not only this, but the fact where Gammarus sp. shows interaction with the multiple trophic levels within water ecosystem, functioning as predator, prey, detrivore, herbivore or as shredder, makes it inevitable that the genus has a wider span of participation in the aquatic ecosystem. This underlines its importance to ecosystem disturbances and pollutants wherein it acts as a bioindicator species for determining the health of the concerned ecosystem where they reside (Kunz et al., 2010). In the present research, the impact of micro-pollutants upon Gammarus pulex behaviors of predator avoidance, mating, feeding activity, development, reproduction, population structure has been focused upon, alongside determination of micro-pollutant impact on predator-prey, host-parasitic, invasive-native species interaction, for revealing wastewater ecosystem health through evaluation of ecotoxicology endpoints. Wastewater ecosystem has been centered upon in the present research due to the innumerous negative impacts which water effluents and pollutants are causing with the concentration of micro-pollutants increasing in the wastewater bodies in the present era (Escher, et al., 2014). Industrialization, and the growth in pharmaceuticals have caused an upsurge in micro-pollutants as found in wastewater effluents, requiring state of the art wastewater treatment facilities and research considering the same, as these effluents containing micro-pollutants have the potency of severely disrupting our aquatic ecosystem at large. Health-monitoring of aquatic ecosystem is thereby crucial, requiring the help of amphipoid genus studies, which serve as potent bio-indicators to assess ecosystem health and to identify problems associated with pollutants for remediation and future amelioration (Escher, et al., 2014).
The micropollutants comprise of algal toxins, chemical pesticides, industrial effluents (slaughterhouses, pharmacies, hospital wastes, textile and paper industries), wastewater influents, VOC or volatile-organic-compounds like chlorinated solvents, PAHs or polynuclear-aromatic-hydrocarbons, MIB, ECDs or endocrine-disruptors, NDMA or 1,4-dioxane, -N-nitrosodimethylamine, or MTBE (methyl-tertiary-butyl-ether) (Margot et al., 2015). The massive production of micropollutants which is aggravated further by the low-removal-capacity of the same even after waste-water treatment in the conventional WWTP (Wastewater-Treatment-Plant) have caused the micropollutants to be high in persistence and they remain in an alarming concentration in wastewater effluents (Eggen et al., 2014). This results them to get accumulated in the soils, followed by biomagnifications of the same in plant bodies (shoot and/ root) or in living organisms feeding on plants as one move from one trophic level onto another, aggravating the harmful impacts of micro-pollutants and heavy metals which has been documented in several research studies and literary texts in the recent decade. Micropollutants if present in alarmingly higher concentrations act as the endocrine disruptors of the aquatic organisms. Further on, the PAHs lead to carcinogenic effects in case of humans (Krupadam et al., 2010). Furthermore, the presence of antibiotics as micropollutants have increased the bacterial resistance towards antibiotics, leading to the influx of drug-resistant microbes in the environment. These potent harms caused by the alarming increase in micropollutant concentration in wastewater ecosystems, is the prime reason behind developing the present research. The present study thus aims to underline how micropollutant impact upon the ecosystem health using the amphipod species of Gammarus pulex and its behavior studies as a potent bioindicator.
Gammarus species and their presence is of utmost importance in the aquatic ecosystem, as macroinvertebrate feeding comprises of a significant rate-limiting stage during aquatic detritus processing and degradation (A Abo-Taleb et al., 2020). However, the increase in contaminant or micropollutant concentration within wastewater ecosystem, reduces detritus processing by the decrease in microbial conditioning. Also, detritus processing may get reduced by abundance in detrivores and their feeding activity (Harmon, & Wiley, 2010). There exist several behavioral activities of Gammarus pulex species, one of which consists of feeding-activity which gets affected and impacted by the level of environmental pollution in the aquatic environment, in terms of micropollutant concentration influx in the wastewater ecosystems. Reduction in Gammarus pulex feeding rate as induced by the presence of toxicants, may cause significant decrease in growth, fecundity, sizes and survival of Gammarus pulex population living in such wastewaters (Brix et al., 2011). Supporting this Kunz et al., (2010), further opined that this may affect the community structure within the wastewater ecosystem at large.
In ecotoxicology research, behavioral parameters are often used as these bear certain advantages, namely - a shorter response time resulting in early-warning symptoms, sensitivity for the neuromuscular toxins, ecological relevance, non-invasiveness and time-bound analysis of data (Gerhardt, 2011). The behavior changes in Gammarus pulex are thereby considered as important bioindicators concerning the wastewater ecosystem's health. This is since the behavior of these organisms is driven by biochemical processes and reflect individual-level fitness of the species or population-level fitness, like that of alterations in species abundance amongst the diverse ecosystems.
During the past decades, monitoring of toxic wastes, specifically the micropollutants present as water effluents has been considered an issue which demands increased concern considering the micropollutants like pharmaceuticals, drugs, cosmetics, industrial wastes, hospital wastes or chemical and biochemical wastes. This is due to their persistent occurrence in groundwater bodies, surface water, treated effluents and in raw wastewater, aggravated by their potent harmful nature to the ecosystem, its inhabitants, community structure and to human health. These contaminant concentrations vary over time taking in consideration, durations on year, month, week or day basis. These pollutants are also commonly found within raw wastewater of petrochemical treatment plants, obtained from diverse sources of production within industrial pole and characterized by a diverse range in pollutant type and their harmful impacts. The report suggested by Verlicchi et al., (2011), is noteworthy in this context, where in Port Marghera, a petrochemical site near Venice, it is mandatory for the WWTP established to strictly abide by authorized limits of micropollutant occurrence concerning 10 major micropollutants of arsenic, cyanides, mercury, cadmium, lead, chloride based organic pesticides, tributyltin, PCB (Polychlorinated biphenyls), hexachlorobenzene, PAHs, and dioxins; commonly referred as "the forbidden 10 substances" within treated wastewater effluent.
The concentration and persistency of micro-pollutants in wastewater effluents even post treatment by conventional WWTP, is an alarming issue, which created potent harm not only to human health, but also to the community health, and health of the ecosystem at large (Eggen et al., 2014). Thus, the rationale of the present study lies in monitoring the impact of such micropollutants on ecosystem health, to understand the implications and measure the potency of harm, which micropollutants are causing in the wastewater ecosystem and to the aquatic communities. The gammarids are widely investigated upon, and amongst the most common species which are mostly studied upon are namely - Gammarus pulex and Gammarus fossarum, both being important species within aquatic ecosystems. The study has focused on considering the behavior of Gammarus pulex, as an impact of micropollutants ion wastewater effluents, it being sensitive to the pollutants as well as other ecological disturbances, thereby acting as valuable indicators to measure and monitor ecosystem health of the wastewater bodies. In several previous research they have been selected in several exposure scenarios like ex situ, in situ, pulsed exposure of through sediments (Gerhardt, 2011). According to Love (2018), for the last decade, in wastewater ecotoxicology, Gammarus pulex's behavior endpoints has been considered together with that of crayfishes, fishes, copepods and other species of gammarids. In the specific case of the Gammarus pulex species, through tests on behavior alteration, researchers can easily measure specific endpoints in relation to population density, population structure, as well as intra or inter specific interactions. The endpoints can thus be considered as sensitive indicators for wastewater ecosystem related chemical stressors, during biomonitoring practices, which in turn can then be utilized for quantitative measurement of species behaviors upon recording the same, like that of filter-feeding, ventilation, locomotion, net-spinning or grazing.
The present study is focused on the aim "to measure and identify the impact which micropollutants present in wastewater effluents may cause, by analyzing the behavior response of Gammarus pulex, as a bioindicator species."
Based on the research aim, certain research objectives have been developed to conduct the research in the proper direction. These have been presented below:
(i) To conduct a feeding-inhibition study on Gammarus pulex, for determining ecotoxicological impact of micropollutants in wastewater (ii) To conduct biota-analysis for identifying micropollutant quantification within whole-body of Gammarus pulex (iii) To conduct behavior analysis specific to anti-predator behavior, swimming endurance alongside other endpoints, through pollution-bioassay
The Research questions devised for conducting the research in the proper direction are as follows: Q1) How can micropollutant ecotoxicological impact be analyzed by feeding-rate-inhibition experimentation studies on Gammarus pulex? Q2) How can micropollutant quantification be determined through biota-analysis using whole-body extracts of the Gammarus pulex species? Q3) How can other behaviors of Gammarus pulex like swimming endurance, anti-predator behavior, be credible for assessing wastewater ecosystem health?
The micropollutants are anthropogenic chemicals occurring in aquatic environments above the natural level caused by human activities where their concentrations remain in microgram/Liter or in trace levels (Figure 1 below). Hence, these pollutants are defined by their occurrence and anthropogenic origin. According to Schwarzenbach et al., (2010), there are several chemicals which fall within the category of micropollutants, accounting to thousands in number. These can comprise of either synthetic man-made chemicals namely - halogenated molecules like the fluoride surfactants, or of compounds occurring naturally namely antibiotics of natural origin like penicillin, estrogens. Micropollutants thus originate from diverse sources comprising of agriculture, traffic network, household source, industries, slaughterhouses, etc., to enter the effluent flow within water bodies following different entry pathways. Based on different sources, the micropollutants get transferred as point source of pollution or as diffuse sources of pollution (Micropollutant - an overview | ScienceDirect Topics, 2016). Micropollutants originated from sources like agricultural waste or land use waste suggest diffuse pollution sources, while point sources refer to examples like micropollutants in wastewater effluents, or WWTPs' which has been focused upon in the present study.Figure 1. The figure depicts the boxplots of pollutant concentration ranges differentiating between micro and macro pollutants. DOC: Dissolved-organic-carbon, TP: Total-phosphorus content (Source: Micropollutant - an overview | ScienceDirect Topics, 2016) From the above figure differences in pollutant concentrations can be seen, where the blue grey boxes signify the micropollutants, whereas green-grey boxes refer to as macro pollutants, with heavy metals in cream-colored boxes appearing in between them. The red line drawn in the center refers to such concentrations used for discriminating micropollutants from the other categories of pollutants. Furthermore, the size of the box denotes to 1st and 3rd quartiles and whiskers extend towards the data point, which is the most extreme, with the circles denoting the outliers. According to Margot et al., (2015), the massive micropollutant production coupled with their lower removal-capacity from the aquatic bodies through traditional WWTPs have caused a massive upsurge of micropollutants in the aquatic bodies as well as a higher persistence. Thus, the micropollutants have started accumulating in harmful concentrations within soil, plant bodies, bodies of living organisms via means of biomagnification, leading to numerous documented harmful effects upon human health, aquatic communities and ecosystem health (Das et al., 2017). This has been supported by Vilela et al., (2018), suggesting that micropollutants have significant impact as endocrine disruptors upon the aquatic animals and marine organisms. This is further confirmed by the research documented by Hirai, et al., (2011), suggesting that the PAHs produce carcinogenic impacts in the humans. The studies of Scheurer, et al., (2015), suggest that higher presence of micropollutants in form of antibiotics, have resulted in bacterial population to develop resistance a wide range of antibiotics, creating drug-resistant bacterial population, occurring as a potent threat to the medical world.
According to Abo-Taleb et al., (2020), the Gammarids' presence in the aquatic ecosystem, and freshwater bodies is essential as the behavior of macroinvertebrate-feeding is considered as a rate-limiting-stage in aquatic detritus processing. Out of the many exhibited behavioral responses of the Gammarids species like Gammarus pulex, feeding activity gets affected by contaminants present in the aquatic environment. Changes observed in their other behaviors like ventilation behavior or locomotion behavior can be reversible and compensatory adaptive responses of the species towards micropollutants which may in turn lead to mitigation of significant overt effects (for example direct response in behavior after stress perception). The irreversible toxicant effect on the expression or behavioral mechanism of the species are also considered as important observational effects post toxicokinetic procedures have been initiated (Ashauer et al., 2012). Cannibalism and intraguild predation, is often exhibited in several amphipoid species including Gammarus pulex, wherein the data suggest that cannibalism is highly common a behavior that what researches noted previously (Lewis et al., 2010). According to Pellan et al., (2016), such "foraging-plasticity" is often linked for the species success in terms of persistence in invading and colonizing aquatic ecosystems which are disturbance prone by nature. Hence, G. pulex 's ecological value in the aquatic ecosystems may exceed to their role as environmental shredder in leaf-material processing or to their role as prey for several species of fishes in the aquatic bodies (Schneider et al., 2015).
The use of G. pulex and its behavioral parameters has several advantages in ecotoxicology-based studies. The shorter response times, sensitivity for the neuromuscular toxins, ecological relevance, non-invasiveness, time-dependent analysis of data are all such significant advantages (Love et al., 2020). Behavior changes of G. pulex thus is often used as potent ecosystem health indicators as they are dependent upon biochemical processes, also reflecting upon fitness (of an individual organism or population level). The behavioral responses are of similar efficiency and sensitivity as the physiological and biochemical responses and owing to their indestructibility, a long-term and continual process of monitoring remains possible to conduct (Kunz et al., 2010). Micropollutant exposure causing impacts in behavior of G. pulex, is hence considered to be significant in behavioral science and in the arena of behavioral ecotoxicology.
In the branch of aquatic ecotoxicology, such behavioral endpoints and their application for crayfishes, fishes, gammarids or copepods have been commonly practiced over the past decade (Gerhardt et al., 2011). The tests on behavior alteration on gammarids allow measurement of several endpoints corresponding to population density, population structure, inter-specific-interaction and intra-specific-interaction. The endpoints act as sensitive chemical stressor-based indicators during biomonitoring procedures (Feckler et al., 2012). This can be further used for recording behaviors quantitatively, like the behaviors concerned with - grazing, ventilation, filter-feeding, locomotion and net spinning. According to Ahlgren et al., (2011), G. pulex are species which are capable of locating their food and in detecting their predators, via chemical cues obtained from the predators like fishes and from injured preys. Furthermore, Harlıoğlu, & Farhadi (2018), opines that, behavior changes like hiding when predators are sensed, or avoiding chemical stresses from micropollutant pollution are highly essential for the survival of this species. Reproductive behaviors of G pulex, in terms of females and males of the species possessing the ability of detecting each other can also be observed. These pairs are then observed to form pre-copulatory pairs in the pre-mating stage, displaying guarding behavior during that period (Franceschi et al., 2010).
Apart from the aforementioned literary evidences supporting impact of micropollutants on G. pulex behavior involving - feeding, mortality, mating and reproduction, several other literary works also exist which address impact of micropollutants and pollution of aquatic environment upon the G. pulex species population size, stress responses, endocrine system, bioenergetics, or neural system (Love et al., 2020). Amongst them few literatures address G pulex 's endocrine disruption, wherein the investigated key endpoints include - size, structure, sex ratio of adults, frequency-length distribution, precopula pair number, ovegerous female number, and the secondary-sexual characters (Langston, 2020). Also studies of Leroy, Haubruge et al., (2010), suggest that in the recent times diverse biomarker-based studies using biomarkers like hsp (heat-shock-protein) or Vtg (vitellogenin) have become common for investigating the process of endocrine disruption within the concerned species. Furthermore, the use of biomarkers has been conducted in the recent times for assessment of G pulex stress responses, exoskeleton integrity, oxidative stress responses and neurotoxicity within them (Elwahaishi, 2012). Also, the aforementioned toxicity endpoint studies, are being used rampantly in the present research for addressing impact at population and individual level on G. pulex and this has been coupled with researches on composition and structure of communities of the respective genus. According to Truhlar, & Aldridge (2015), the species Dikerogammarus villosus, is considerably well known as an invader species and is at present the most prevailing species of gammarid found in the aquatic ecosystems of Southern Germany. The field study results suggest that the invasive species D. villosus have been successful in replacing the G. pulex species in certain spans of Danube river flower in parts of Germany. These shifts in the composition of species, as such may also help in explaining micropollutant based long-term impact upon community structure of certain micropollutant infested waters, and in explaining the differences in species sensitivity observed in respect to these micropollutants.
Thus, in the present section, through the present literature review of the modern studies, an initiative to address the prominent research objectives has been undertaken, to later carry out the research analysis process and the process of discussion, with clarity and validity.
Among the various approaches that can be adopted by research studies, primary and secondary researches are the main approaches. The former chiefly entails the collection of first hand data from the field while he latter involves the usage of data that is already collected by other researchers (Largan & Morris, 2019). Qualitative and quantitative research designs are the major paths that are taken by these approaches. As qualitative research methods involves themes and words, the quantitative design primarily involve statistical data. This research study adopts a secondary research methodology where data is collected from the databases where they have been posted by other researchers. This methodology was also chosen based on its efficiency and the fact that is cheap compared to primary research. In this study, papers were systematically reviewed to collect data from the selected papers that would be useful in answering the research questions. The databases that were searched for relevant literature included PubMed, ECOTOX-EPA, Ecotoxicology-Springer and Google Scholar. The databases were chosen on the rationale that they have broad content that is relevant to the topic of study.
Information were gotten from the existing literature. The sources were obtained from various databases. The chosen databases included PubMed, ECOTOX-EPA, Ecotoxicology-Springer and Google Scholar.
Detailed content analysis was conducted, considering only the research works and academic literatures, which have been conducted over a period of the last 10 years, so as to maintain data credibility and validity. A systematic review has been considered as the major approach in the secondary research-based study. This had aided in designing the present study, in a holistic way via the use of quantitative data pool on the topic of impact of micropollutants on behavior response of Gammarus pulex. Furthermore, priority has been put forth on selecting observation based primary research for enhancement of in-depth analysis and research efficiency. This would hence help in conducting proper assessment of the research topic in question, supporting it with relevant observational studies, proper result analysis and appropriate conclusions.
The participants included 10 individuals that voluntarily agreed to assist in the identification and synthesis of relevant studies through systematic reviews.
The search strategy which has been adopted in the present study, comprises of systematic web-searches which has been conducted in a holistic manner for the procurement of appropriate and relevant data, from valid platforms like peer-reviewed journals, international journal based research, government websites, all published within last 10 years such that the data so collected and taken in consideration remains relevant. Also, only those studies and literary works have been considered where the research topic of impact upon Gammarus pulex behavior response upon impact by micropollutants, has been focused upon, in a major part of the research or literary body. Furthermore, profile-based research data have been taken in consideration for the present study, to assess the research topic in detail focusing on how the behavioral response of Gammarus pulex, gets impacted by the micropollutant levels in wastewater effluents.
While searching the databases for relevant literature, Keywords were used to search for relevant studies from the chosen databases. The keywords used were inclusive of micro-pollutants, wastewater effluent, behavior response, chemical and industrial water pollutants, wastewater, and aquatic ecosystem, Ecotoxicology, and Gammarus pulex. The research topic was also used to identify relevant studies for review. Boolean search terms and operators were used in the survey, which helped minimize the appropriate search for the research topic and question. The abstraction of different studies was also limited by a combination of two or more databases.
The role of reviews in science remains impeccable and inevitable. Studies can adopt various kinds of reviews that have been noted to include critical reviews that demonstrate an extensive research of literature and critical evaluation of the quality of the same resulting in hypothesis or model. Besides, research can adopt mapping review that maps out and categorizes existing literature from which to commission further reviews. Meta-analysis, another possible approach, statistically combines the results of quantitative studies to offer precise effect of the results. Also, mixed studies review has been discussed by many scholars as a combination of methods such as qualitative and quantitative. Qualitative systematic review integrates and compares the findings from qualitative studies seeking themes. Rapid review and systematic review are other forms whereby the latter seeks to systematically search for, appraise, and synthesize research evidence to answer a pre-set research question.
In the present research, a systematic review approach towards conducting the research forward has been considered based on its various advantages, these have been noted to be inclusive of delivering a clear and comprehensive overview of available evidence on a given topic of study. Besides, the methodology design identifies research gaps in the current understanding of a field such as the one chosen for thus particular dissertation. It highlights methodological concerns in research studies that are significant to the improvement of future works in the chosen topic of study, and lastly, systematic reviews help in the identification of questions for which the present evidence answers and provides a rationale for further studies. These amongst other reasons formed the rationale for the choice of the research design.
During the literature search, review and research journals and literary texts of international repute have been considered, together with internationally acclaimed journals, and peer-reviewed manuscripts for procurement of secondary data, well extracted from reliable and valid sources. Systemic review has been chosen based on the rationale that the data so procured have then been tallied with other procured articles and research work, drawing a comparison amongst them for eventually selecting some, and rejecting the other data sources. They are then jotted in word documents, with routine assessments conducted upon them. Also, to properly assess the procured research and data quality standards, the CASP RCT protocol for systematic reviews have been considered and followed as base guideline (Zeng, et al., 2015). This has further aided in systematically selecting proper and efficient data from a data pool of literary texts and documents, comprising of trail-based experiments, stressor-based experiments, endpoint based ecotoxicology researches for assessing and evaluating the applicability of the collected research data.
In the present research, data has been collected from several search-engines, one primary search engine which has been taken in present study is Google-Scholar, from which several research and review based articles have been selected alongside books, literary documents using effective key words for the conduction of the search. The publication year, author credibility and journal repute, IUCN accredited journals are other search-based criterions which have been considered for data collection. Furthermore, other databases of repute namely, ECOTOX - EPA, which is an ecotoxicology-based database, as well as PubMed Database has also been taken in consideration for carrying out effective data search ("ECOTOX | Home", 2020). Furthermore, the journal of Ecotoxicology-Springer has been considered for data procurement and search.
In the present study, the MESH terms which were used for the search procedure, were selected such that they bear a close association of the present research topic, on how micropollutants create an impact upon the behavior response of the Gammarid species Gammarus pulex. Thus, the selected MESH terms were: Gammarus pulex, micropollutant, wastewater, aquatic ecosystem, behavior response, ecotoxicology, wastewater effluent, Gammarus, gammerids, chemical pollutants, industrial pollutants.
Gammarus pulex, refers to a commonly found amphipod species of crustaceans, who commonly inhabit the freshwater bodies of Northern hemisphere. They are grey colored crustaceans, growing in size to 0.83 inches in length and are widely used in ecotoxicology-based research, their biology having been thoroughly studied and well-known (Kunz et al., 2010). Micropollutant refers to anthropogenic chemical substances, produced from human activities occurring in aquatic bodies, above the natural level but remains in trace-level concentration in µg/Lt range. Wastewater refers to water which has been polluted or contaminated through human usage and comprises of water which has already been used in domestic purposes, or in commercial, industrial, agricultural work. Water from sewer inflow, storm water or surface runoff all refer to wastewater (Eggen et al., 2014). Aquatic ecosystem refers to water-body ecosystems. The major two types are the freshwater and the marine ecosystem. Behavior response entails to the actions, interactions of a species as behavior, to the external or internal cues, as responses. Ecotoxicology refers to study of toxic chemicals and their efficacies upon the biological organisms at community level, population level, ecosystem level and biosphere level. Wastewater effluent denotes to the out-flow of gas or water to that of a natural water body, from WWTPs, industrial outfalls, sewer pipes. Gammarus refers to the amphiphoid genus belonging to the Gammaridae family and comprise of crustaceans. With more than 200 diverse species within the genus, the genus Gammarus is considered to contain the highest species-richness amongst the crustaceans. Gammaridae, refer to an amphipod family, with wide geographical distribution specially in Northern Hemisphere and Eurasia, inhabiting both the marine and freshwater ecosystems (Kunz et al., 2010). Chemical pollutants refer to chemical substances entering the ecosystem via means of industrial, human, agricultural activities posing potent or immediate hazards to the organisms. Heavy metals like mercury, arsenic, lead, nickel, zinc are some chemical pollutants who in excessive amounts cause severe biomagnification and affects health of humans and other organisms (Patnaik, 2017). Industrial water pollutants refer to wastes discharged from industries and city sewage onto water bodies generated through industrial and manufacturing procedures. These hazardous wastes are often detrimental to human health and to the biota.
In the present study the search engines, journals, and databases taken in consideration are PubMed, ECOTOX-EPA, Ecotoxicology-Springer and Google Scholar. These databases and search-engines have been systematically searched using keywords, with preference provided to recent data published within the last 10 years, trial based studies, stressor studies, end-point based researches, and biomarker study based researches wherein behavior responses of Gammarus pulex have been analyzed in details, with the research results and conclusion staying in clear alignment with the objectives and topic of research. However, the abstracts from thesis have not been considered as there was a lack of detailed information on research methods adopted and results obtained from the same. Furthermore, keyword-based search was conducted using keywords like - Gammarus pulex, micropollutant, wastewater, aquatic ecosystem, behavior response, ecotoxicology, wastewater effluent, Gammarus, Gammarids, chemical pollutants, industrial pollutants. From Google scholar-based search 141 outcomes resulted, from which after an in-depth screening following the CASP SR protocol, 1 outcome was selected. From the ECOTOX-EPA database, 153 outcomes were obtained from which 3 outcomes were finally selected and the rest rejected. From the Ecotoxicology-Springer Journal 129 outcomes were obtained from which 1 outcome was selected for the present research study, based on case proximity and closeness to the present research topic on identifying behavioral response of G. pulex species as impact of micropollutants in wastewater effluent bodies. Finally, from PUBMED search 121 outcomes were generated, out of which 3 outcomes were finally selected for the present study.
All the data and manuscripts selected for the present research went through processes like individual-crosschecks, screening, for decreasing the chances of duplication of manuscripts and to select only the most relevant articles or research which remain updated within the 10-year time period considered. This was followed by a second follow-up which traversed through the research data sets, selecting them on basis of certain inclusion criteria, or rejecting them on basis of exclusion criteria as discussed in the underlying sections of the present study.
The literature search criteria focused upon the CASP - SR protocol, for selecting certain studies for the present research, while other studies were rejected. The inclusion criteria considered for selection of specific data and manuscripts for the present research are given below: 1. Synchrony between topic aim, methodology and results obtained was considered mandatory and such studies were included for the research. 2. Research studies which showed confirmed impacts of micropollutants on behavioral response of G. pulex were considered fit for inclusion. 3. Studies where behavior changes of G. pulex, were clearly demonstrated were included. 4. Studies indicating irreversible toxicant effect on G. pulex 's expression were included in the present research. 5. Studies on - sensitivity of the species for neuromuscular toxins and on ecological relevance, were considered. 6. The studies focused upon measurement of several endpoints in terms of - population density, population structure, inter-specific-interaction and intra-specific-interaction, and stressor studies were also prioritized over other research. 7. Biomarker based studies for determining stress responses of G. pulex were also prioritized. 8. Studies date ten years and below 9. Peer reviewed articles, journals and books 10. Studies that contained most of the keywords that were used in the search 11. Studies relevant to the topic of study 12. Studies published in English
Studies that were excluded from the systematic review were those that did not meet the set inclusion criteria and thus whose features aligned with the designed exclusion criteria. The exclusion criteria for the papers were as outlined below. Studies that were published in other languages other than English. Studies that were dated more than ten years from the time of retrieval. Studies that lacked most of the key words that were used in the literature search. Studies that lacked a Synchrony between topic aim, methodology and results obtained Studies that were irrelevant to the topic of study Non peer reviewed articles, journals and books and studies that were not full text.
In the data analysis plan, the research questions were first to be answered. Also, the evidence was divided to liken the sentiments from the dissimilar researchers. The study used the thematic data analysis that allowed for the identification conjoint leitmotifs (Braun & Clarke, 2012). The chief step in the scrutiny plot intricate publicizing the data unruffled from the appraisals (Vaismoradi et al., 2016). Afterwards, the coding of the facts was conducted through the highlight of sectors of the information in raising codes and in explaining the evidence. After that, themes were generated by identifying patterns among the codes identified. The accuracy of the themes was then validated through a review of the same (Vaismoradi et al., 2016). The progressions enabled the investigator to recognize omissions and inclusions. Besides, conceivable vicissitudes to make the themes work better were articulated (Vaismoradi et al., 2016). The next step in the data analysis plan was defining and naming the themes. Describing the refrains convoluted articulating individual theme's particular connotation moreover reckoning how the explicit themes assisted comprehend the material composed from the appraisals. Designation of the themes involved conveying concise and modest to understand each theme's names (Braun & Clarke, 2012).
Systematic reviews are occasionally read and cited in documents that influence educational policy and practice. Consequently, ethical considerations linked with what and how systematic reviews are produced and used are a major factor that should be considered while conducting a secondary qualitative research. It is of utmost importance for systematic reviews to reflexively engage with a variety of ethical issues that are lined concerns of interest and issues of voice and representation. Systematic reviews use publicly accessible documents as evidence thus seldom seek institutional approval before undertaking. In practicing informed subjectivity and reflexivity, researchers need an effective epistemological orientation such as post-positivist, interpretive, and participatory. For a participatory systematic review, ethical considerations should cater for the participants as well as the general public.
According to Munhall (1988), the interaction between researchers and participants is ethically challenging in various aspects. Considering that, conducting qualitative research requires adherence to several ethics of the research. The author adds that the formulation of an ethical guideline before qualitative research is of utmost importance. Ethical challenges are faced by designing a research study on the reporting stage (Raudonis, 1992). These include anonymity, confidentiality, informed consent, researchers' potential impact on the participants, and the participants' impact on the researcher. For this study, ethics was put into consideration. For instance, it was ensured that the person taking parts in the evaluation were fully informed about the research (Munhall, 1988). The participants were made aware of the purpose of the project, how the results where be used, possible impacts of participation in the study, and who would have access to the collected information (James & Platzer, 1999). This ensured adherence to the ethics of informed consent. By observing this, the participants made an informed decision on whether they would participate in the research or not.
Another ethical consideration during the research was being voluntary participation (James & Platzer, 1999). The participants were free from coercion. They were free to withdraw their participation at any instances without harming their involvement in future studies or the same study. It was the participants' right to leave a study at any time; thus, no pressure was placed on the participants who choose not to continue (Munhall, 1988). Also, in the study's scope, it was ensured that there was no harm either psychologically or physically. This can be in the form of pain, stress, anxiety, diminishing self-esteem, as well as invasion of privacy (James & Platzer, 1999). The study ensured that the participants were not harmed in any way. They were also ensured of confidentiality. Identifying information of the participants was not to be disclosed. They were to be excluded from any reports or published documents (Raudonis, 1992). Besides, anonymity was ensured that a stricter form of confidentiality is adhered to. Lastly, it was ensured that only relevant components are assessed. It only be focused on the intentions of the study and what data gathered data was to be used for (James & Platzer, 1999).
From a search of the literature, the following studies were identified for review. Álvarez-Ruiz, R. and Picó, Y., 2020. Analysis of emerging and related pollutants in aquatic biota. Trends in Environmental Analytical Chemistry, 25, p.e00082. Love, A., 2018. The sub-lethal impacts of wastewater effluent and its components on Gammarus pulex (Doctoral dissertation, University of Portsmouth). Könemann, S., Müller, Y., Tschentscher, D., Krauss, M., Inostroza, P. A., Brückner, I., Pinnekamp, J., Schiwy, S., & Hollert, H. 2019. Combination of In Situ Feeding Rate Experiments and Chemical Body Burden Analysis to Assess the Influence of Micropollutants in Wastewater on Gammarus pulex. International journal of environmental research and public health, 16(5), 883. https://doi.org/10.3390/ijerph16050883 Kunz, P.Y., Kienle, C. and Gerhardt, A., 2010. Gammarus spp. in aquatic ecotoxicology and water quality assessment: toward integrated multilevel tests. In Reviews of Environmental Contamination and Toxicology Volume 205 (pp. 1-76). Springer, New York, NY. https://www.researchgate.net/deref/http%3A%2F%2Fdx.doi.org%2F10.1007%2F978-1-4419-5623-1_1?_sg%5B0%5D=eOEKh9G77SZ5zmEk7-R-U3Y2fN5y4RmTg4MDI1ZuLKFzCIroZKW9PBiWy9zjKb_cI1xVooxxGdvxyiYb_rnGab29zg.L0_O2yFVsj8Hclfn0VnAJxbZ4xqQeuXJ-0QIFOotfhvD0D4P5P29Za-lghIy4dMbq4t8-HmikHp8RX-VVsL8ug Tatar, S., Cikcikoglu Yildirim, N., Serdar, O., Yildirim, N. and Ogedey, A., 2018. The using of Gammarus pulex as a biomonitor in ecological risk assessment of secondary effluent from municipal wastewater treatment plant in Tunceli, Turkey. Human and Ecological Risk Assessment: An International Journal, 24(3), pp.819-829. Vellinger .C., Felten .V., Sornom .P., Rousselle .P., Beisel .J., Polatera. P. U. 2012. Behavioural and Physiological Responses of Gammarus pulex Exposed to Cadmium and Arsenate at Three Temperatures: Individual and Combined Effects. https://doi.org/10.1371/journal.pone.0039153 Windisch, U., Springer, F. and Stahl, T., 2020. Freshwater amphipods (Gammarus pulex/fossarum) and brown trout as bioindicators for PFC contamination with regard to the aquatic ecological status of a small stream. Environmental Sciences Europe, 32(1), pp.1-14. Zubrod, J.P., 2016. Impact of fungicides on an aquatic decomposer-detritivore system (Doctoral dissertation, Universität Koblenz-Landau).
The Caldwell appraisal tool was applied in the critical appraisal of the identified papers whereby it offered a list of queries against which both the qualitative and quantitative documents were appraised. The dissimilarity between the studies was broad as others were termed to probable, investigative studies, mixed-method studies, and evocative studies, correlational studies, and reflective studies. For this precise investigation study, a mixed-method framework was applied (Caldwell et al., 2011). Chosen studies were disparagingly assessed by the eighteen interrogations as nominated by the Caldwell framework assessment tool. The results from the critical appraisal of the studies are summarized in the table below.
Konneman et al. in their study explored the combination of In situ feeding rate experiments and chemical body burden analysis to assess the influence of micro-pollutants in waste water Gammarus pulex (2019). This study answered the research question of how micro pollutant ecotoxicological impact be analyzed by feeding-rate-inhibition experimentation studies on Gammarus pulex. The study noted that wastewater discharge is one of the main sources of the micro-pollutants in the aquatic ecosystem. The study used a Quick Easy Cheap Effective Rugged and Safe (QuEChERS) multi-residue method to quantify the internal concentrations in collected gammarids to investigate using in situ feeding experiments with Gammarus pulex and body burden analysis of the frequently detected micropollutants. The study detected no significant acute effects when the up and downstream feeding rates of Gammarus pulex were compared for seven days relative to the WasteWater Treatment Plants (WWTPs). However, slight trend of increased feeding rate downstream of the WWTP Aachen-Soers was observed from the study by Konneman et al., (2019). According to Konneman et al., (2019), chemical load from the WWTP was too low to lead to clear acute effects on Gammarus pulex. Some micropollutants in the wastewater altered the microbial community on its leaves increasing the feeding rates of Gammarus pulex. When the internal concentrations of the pollutants were chemically analyzed on the tissues of the collected Gammarids, a potential risk of chronic effects was noted (Konneman et al., 2019). The study by the authors has shown that a combination of acute testing and measurement of the internal concentrations of micropollutants resulting in chronic effects can be effectively used to investigate aquatic environments.
The second study that was identified from the literature search was an analysis of the emerging related pollutants in aquatic biota (Alvarez and Pico, 2020). On synthesizing evidence from the paper systematically, the paper effectively answered the research questions and assisted in meeting the aims and the objectives of the study. The study noted that aquatic environments receive both the direct and indirect discharges of emerging pollutants from all sectors. Consequently, the aquatic biota was noted in the paper to be susceptible to the exposition, bioaccumulation and biomagnification of the emerging pollutants (Alvarez and Pico, 2020). The authors also noted that multi-residue analysis of the aquatic environment is difficult due to the distinct pollutants as well as the complexity of the matrices. The study notes that water bodies are an ecosystem with a significant value habituating various flora and fauna, thus, pollutants affect it diversely. The invertebrate Gammarus Pulex is not an exception of this (Alvarez and Pico, 2020). Besides, the researchers note that the micro pollutants are ubiquitous anthropogenic compounds of the environmental concern that affect the aquatic biota and water systems. They also pose a risk to humans when contaminated biota is consumed (Alvarez and Pico, 2020). The study not only analyses the effect that micro-pollutants pose on Gammarus pulex but aquatic biota. It was thus helpful to the study following the assessments of the strengths and the weakness that he studies presented with. In a different study that explored the sub-lethal impacts of wastewater effluent and its components on Gammarus pulex, Love (2018), states that there has been a dramatic upsurge in the prevalence of pharmaceuticals and personal care products in lotic habitats causing contaminants to be effluent from waste treatment works. The study explores the impact that the PPCPs have on the aquatic biota (Love, 2018). The research aimed to evaluate the effects of the micro-pollutants on the important freshwater invertebrate, Gammarus pulex, through field and laboratory observations. The study demonstrates the effect of micro-pollutants on the energy content in Gammarus pulex. Also, the study has investigated and detailed the effect that micro-pollutants have on the lipid reserves at higher concentrations. This ecotoxicological research focused on the higher toxic concentrations relevant to the environmental situation. The research studied behavioral indicators that were reported to be very sensitive to the micro-pollutants and contaminants. The research used the invertebrate behavior as a biomarker of WwTW effluent. The findings indicated an intial spurt of activity upon exposure to Fullerton WwTW effluent (Love, 2018). Due to the very distinct mode of action of the micro-pollutants, the effects differed with the micro-pollutants themselves. Feeding rates were noted to be inversely related to activity (Love, 2018; Konneman et al. 2019). Besides, review of literature by Love showed an increase in feeding rate to cover for increased energy demand as a result of the toxic stress caused by the micro-pollutants (2018). The study indicated a clear disconnect between the pair rate of Gammarus pulex that were exposed to the micro-pollutants that had a negative effect.
Kunz et al, (2011) conducted a study that summarized the vast number of research that had evaluated aquatic ecotoxicology endpoints. Besides, the study examined the suitability of the Gammarus pulex as an invertebrate species for the assessment of the health of the aquatic ecosystems. The study notes that pollutants significantly affect Gammarids behaviors such as mating, predator avoidance, reproduction, and development, feeding activity, population structure besides consequences of pollution on host-parasite, predator-prey, or native-invasive species interactions (Kunz et al., 2011). The study established biochemical and molecular biomarkers that are significant in the assessment of the health of aquatic ecosystems. Unlike other studies that were systematically reviewed in this dissertation, the study by Kunz et al appreciated the structural and functional significance of Gammarus pulex in freshwaters (2011). Just like the study by Konneman et al (2019), this study the same research question as it discussed how macroinvertebrate feeding is a major factor that is significant to the assessment of the health of aquatic ecosystems. In a different paper, Tatar et al (2018) examined the use of Gammarus pulex as a biomonitor in ecological risk assessment of the secondary effluent. The researchers determined the MDA and GSH levels via a spectrophotometer while ELISA kit was used to determine the SOD, CAT and GPX activities in the groups that were exposed to secondary effluents as well as the control experiment. From the experiment, it was notable that the level of MDA in Gammarus pulex varied based on the exposure time to secondary effluents (Tatar et al., 2018). According to the researchers, the GSH and the GSH-PX activities were also noted to increase in the invertebrate that was exposed to the secondary effluent relative to the one that was not exposed to the effluent. In the SE group. CAT activities were found to be lower than the control in Gammarus pulex. the research showed that Gammarus pulex is sensitive and appropriate for the assessment of the effects of micro-pollutants on the aquatic ecosystems following the effects that the micro-pollutants has on the SOD, CAT, GPX activities and GSH and MDA levels in the invertebrate (Tatar et al., 2018).
Cadmium and arsenate are some of the pollutants that affect Gammarus pulex in a broad context (Vellinger et al., 2012). In their study, the researchers explored the various behavioral and physiological responses that Gammarus pulex exhibits when exposed to the stated micro-pollutants. As the study aimed to examine the effects of micro-pollutants on the invertebrate behaviors, the study by Vellinger et al was thus considered as relevant and competent enough for the study, thus the choice. The study noted that the mortality of Gammarus pulex significantly increased in response to the exposure to the micro-pollutants relative to individuals that were not exposed (2012). The study also noted a gradual decrease in haemolymphatic activity in Gammarus pulex that was exposed to single cadmium. In conclusion, micro-pollutants greatly affect Gammarus pulex in adverse ways that include an increase in mortality rates. Windisch et al examined the application of freshwater amphipods such as Gammarus pulex as bioindicators for micro-pollutants contamination regarding the aquatic ecological status (2020). In the study, the researchers noted that micro-pollutants such as per – and polyfluorinated chemicals (PFC), can be detected in Gammarus pulex whereby the highest concentrations are noted in the liver and kidney (Windisch et al., 2020). From the study, the researchers noted that Gammarus pulex was useful as a bioindicators for PFC contamination of streams based on the effects that PFC has on the amphipod. The study by Zubrod (2016) was the last one that was systematically reviewed to draw evidence that would be used to answer the research questions and achieve the pre-set research aims and objectives. The study was exploring the impact of fungicides has on an aquatic decomposer detritivore system (Zubrod, 2016). The researcher noted in his study that fungicides pose a specific risk since they present a negative effect on the microbial decomposers that affects shredders such as Gammarus pulex via waterborne toxicity as well as the diet. The researcher provided a detailed assessment of the ecotoxicological implications of fungicides, which is a micro-pollutant (Zubrod, 2016). The research states that antifungal substances act on biological processes which are highly conserved. Thus, fungicides greatly impact a range of distinct taxonomic groups of aquatic organisms such as Gammarus pulex. This results in potential for toxic effects in shredders when subjected to waterborne exposure towards these micro-pollutants. The study demonstrated an implication in leaf breakdown via feeding of the Gammarus pulex. Fungicides also affect shredders via their diet (Zubrod, 2016). For instance, the accumulation of fungicides on leaf material causes toxic effects when the antifungal substance is co-ingested with the leaf substrate. Fungicides affect fungal leaf decomposer indicating detrimental effects on microbial condition and food quality of leaf material for the shredders like Gammarus pulex. All the studies that were systematically analyzed were relevant and answered the research questions effectively.
The results presented show a significant effect that micro-pollutants have in wastewater effluent on the behavior response of Gammarus pulex. The macroinvertebrate community of organisms should be analyzed tested in future studies. The Gammarus genus is of high significance functionally and structurally in the freshwater ecosystems specifically of Northern Hemispheres Wastewater ecosystem has been centered upon in the present research due to the innumerous negative impacts which water effluents and pollutants are causing with the concentration of micro-pollutants increasing in the wastewater bodies. The micropollutants comprise of algal toxins, chemical pesticides, industrial effluents (slaughterhouses, pharmacies, hospital wastes, textile and paper industries), wastewater influents, VOC or volatile-organic-compounds like chlorinated solvents, PAHs or polynuclear-aromatic-hydrocarbons, MIB, ECDs or endocrine-disruptors, NDMA or 1,4-dioxane,-N-nitrosodimethylamine, or MTBE (methyl-tertiary-butyl-ether). The study aimed aims to underline how micropollutant impact upon the ecosystem health using the amphipod species of Gammarus pulex and its behavior studies as a potent bioindicator. In the first sections of the research an introduction to the topic of research has been provided, followed by an in-depth literature review considering the recent academic works conducted within the last 10 years. A secondary methodology of data collection has been considered most suiting the research, as the research topic requires a wide range of knowledge gained from literary sources of repute, and from acclaimed journals for attaining all the objectives. A PRISMA Flowchart has been provided to throw light upon the literature searches made and the reasons behind why some research has been considered in the present study while others have been ignored. Data has been analyzed and result interpretation has been conducted to be followed by future research plan and a conclusion of all the important facts obtained in the study.
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