**1.1 Salt farming toward a greener future**

Saline farming is based on the cultivation of crops and plant varieties that can tolerate high levels of salinity and temperature. Its idea originated primarily from nature itself and from the growth of naturally salty plants on sandy beaches, tidal areas, saline lands, and other saline-flooded areas [1–4]. The most prominent example of this type of plant is the mangrove plant, also known as the Crimea or Shura, which is heavily distributed on the shores of the Red Sea and the shores of the Arabian Gulf. The Crimea—and this type of plant in general—adapts to

the saline environment in more than one way and mechanism. Salts, which are collected and then disposed or adapted to the saline environment in the place they are found, contain a high degree of salinity [5, 6]. Some researchers have tried to mimic these natural conditions by using saline water to irrigate and grow some plant and crop species. One may succeed in adapting to high salinity and continuing to grow and produce [7]. The first attempt began in 1949, but the expansion of research and scientific experimentation on the cultivation of saline plants began only in the late seventies of the last century. Since then, many scientific institutions, such as Arab research centers, have been actively trying to develop new techniques for salt farming and to develop new varieties of salt-tolerant plants and crops, whether from major food crops such as wheat and barley or from other plants that can be exploited as natural pastures or fodder for livestock [8–10]. However, the concept of saline agriculture is not only to improve the ability of some plants to grow and mature in a harsh saline environment and to irrigate and plant certain plant and crop species with high salinity water but also to use brine or improve some of their properties or specifications, such as sugar concentration in fruit [11–14].

In general, it is possible to confirm that the process of developing saline farming techniques and the production of saline-resistant plant species took two different scientific approaches: the first was the application of genetic engineering technique to genetically and geologically transform traditional plants and transform them from being saline to tolerant. While the second approach is based on the cultivation of saline plants that can tolerate salinity and try to expand their cultivation in the wild and agricultural fields for use as food crops or animal feed or for the production of oilseeds.

The expansion of saline agriculture can be of greater benefit, such as the exploitation of arid and semi-arid lands, which sometimes have abundant amounts of brackish water unsuitable for conventional agriculture. Cultivating some suitable crop types and increasing their production will help to achieve food security [15–17]. Similarly, marginal beaches exposed to tidal movement can also be exploited for feed farming or other plant species that can be used to produce energy (biofuels) or to extract pharmaceuticals or oils [18–20].

Salt farming can also contribute to increasing the efficiency of the use of water resources by conserving potable water or traditional agriculture, which in turn helps to achieve water security and reduce migrations and conflicts resulting from the lack of water, land degradation, and increased drought. In addition, salt farming techniques can also contribute to improving the productive efficiency of some crops or improving the quality and characteristics of certain crops, thereby achieving high economic returns [21–24]. Saline farming can also contribute to mitigating the effects of global warming leading to climate change. This is due to its role in increasing agricultural land and green plant areas that absorb carbon dioxide from the atmosphere [25–29].

As such, salt farming can support traditional agriculture and raise pressure in it, since the requirement of traditional agriculture is to find less water-consuming ways and increase agricultural production to meet the growing demand for food and staple crops. Traditional agricultural activities are the main consumers of drinking water by 70%, followed by industrial activities by 20%, and other activities.

Pibars and Mansour [30] found that severe soil water deficit (SWD) decreased grain yield of winter wheat, while slight SWD throughout the growing season did not reduce grain yield or water productivity. This result indicates that water supply can be reduced somewhat without significant decrease in grain yield. Moreover,

**59**

world [44].

[11–13].

*Performance of Water Desalination and Modern Irrigation Systems for Improving Water…*

investigations conducted by [31, 32] show that deficit irrigation can increase the net farm income. Barley, considered as a tolerant plant [33], occupies large cultivated areas in arid parts of Tunisia. Many experiments have been conducted on barley cultivated in small private farms in southern Tunisia [34] and the results demonstrate the potential of irrigation management practices in reducing the effects of salinity on both yield and soil salinity. In addition, [35] showed that yield reduction under deficit irrigation during the whole growing season was about 5% and 20% of

There are about 295 million hectares of coastal desert land in the world, where about 17% (about 50 million hectares) is level 0% slope lands, it's suitable for irrigated agriculture (in terms of soil type, the land slope, and there is no competition for the other uses), and it's expected that the land will increase the irrigated areas in the desert regions by about 80%. The common regions for such use are the different deltas of some rivers where coastal sediments constitute sedimentary desert lands such as the Nile River (Egypt), Euphrates River (Iraq), and Colorado River (US), where the sedimentary coastal delta is often suffering from secondary salinity

Such spaces can be introduced into economically valuable agricultural production by cultivating halophytes using seawater. The sandy coastal desert along the coast of the Red Sea and the Arabian Gulf, as well as the Indian Ocean and the Gulf of California, are suitable for this type of use, adding additional areas suitable for irrigation by seawater. Many coastal plains of the sea, as well as in the northern coastal plains of Australia and some areas close to population centers or some large cities such as Cairo, Baghdad, Bombay, and Karachi, are also common for such use. This is a great opportunity to invest in the production of animal feed from halophytes, which reduces the pressure on the use of fresh water and available agricul-

It was projected that the desalination technology would provide nuclear power, where it could provide a cheap source of energy for seawater desalination that could be used for agricultural and land reclamation. But to some extent, these techniques are still expensive and require high investment. Therefore, the direct use of seawater in agriculture is an optimistic and great hope for agricultural development along the coastal deserts by growing salinity-rich and economically profitable crops

Some researchers have tried to cultivate traditional crops on seawater such as barley, which can at least complete their life cycle on seawater in temperate climates [40–42]. Furthermore, it has been assumed that breeding programs for such crops can be developed to improve their salinity tolerance and to create mutations of resistance or salinity. But so far, no conventional crops have been found that can produce an acceptable economic yield under irrigation conditions in the sea in the coastal desert climate [43]. Recently, a new approach has been proposed: to attempt to settle or rehabilitate and cultivate saline-loving plants that grow naturally in such conditions for agricultural production and thus can be used as a natural resistance to salinity. Some countries, such as North Africa, have begun using this technique to produce seeds and halophytes using seawater. About 15 years ago, the University of Arizona began field experiments to cultivate halophytes in many parts of the desert

With increased experience and information, experimental plots were increased from 0.5–1 ha to 20–40 ha experimental farms, and different irrigation methods

tural land and reduces overgrazing on relatively few grasslands [38, 39].

*DOI: http://dx.doi.org/10.5772/intechopen.87010*

the total irrigation water was saved.

**1.2 The need to use seawater in irrigation**

problems. Many places suffer from desertification [36, 37].

investigations conducted by [31, 32] show that deficit irrigation can increase the net farm income. Barley, considered as a tolerant plant [33], occupies large cultivated areas in arid parts of Tunisia. Many experiments have been conducted on barley cultivated in small private farms in southern Tunisia [34] and the results demonstrate the potential of irrigation management practices in reducing the effects of salinity on both yield and soil salinity. In addition, [35] showed that yield reduction under deficit irrigation during the whole growing season was about 5% and 20% of the total irrigation water was saved.


**Abstract**

In oil fields CO2-EOR have extravagant interest because of its increasing microscopic sweep efficiency. As a balance and dense solution over a huge range of temperature and pressure, carbon dioxide can improve viscosity reduction and oil swelling because of all partial miscibility with heavy oils and proportion miscibility with light oils, and also carbon dioxide has mutual solubility with water and hydrocarbons that these properties result in increasing microscopic sweep efficiency in mechanism of CO2 injection. However, due to CO2 lower viscosity than water and oil, CO2-EOR efficiency is limited that causes near well-bore conformance issue and mobility contrast problem such as gravity override and fingering. The carbon dioxide philic surfactant blends traditionally includes foam booster, foam stabiliser and foaming surfactant. An integrated propertyperformance analysis of blends of anionic surfactants includes carbon dioxide philic groups such as twin-tailed carbonyl group, propylene-oxide and methyl group of CO2 philic groups under the circumstances of reservoir showed that carbon dioxide philic surfactants can be the mixture of CO2 problems. Here we will underline that modifying the surfactant tail can be a proper path to surpass foaming performance.

**Keywords:** EOR, foam, CO2-philic surfactants


**7. Future direction and research**

Modern technological advancements have permitted the development of surgical simulators that mimic complex operations specific to the anatomical demands of individual patients. By allowing surgeons to rehearse the precise case they

*Perspective Chapter: Simulation in Complex Laparoscopic Digestive Surgery DOI: http://dx.doi.org/10.5772/intechopen.108224*

#### **Figure 4.** *VR application for the preoperative demonstration of 3D liver models [62].*

will be conducting on models that accurately mimic their patients, these patientspecific surgical simulators reach the maximum level of realism. Furthermore, telesurgery is developing into a genuine tool for skilled surgeons to mentor novice surgeons in complex surgical procedures thanks to augmented reality and wireless technology [60].

Three-dimensional printing technology is also evolving with new applications in surgery being developed. For instance, in liver surgery, anatomy data can be extracted from the CT or MRI scans of a patient with a liver tumor and converted into a digital 3D model which can be 3D printed. The final model can show the surgeon the relationship between the tumor and surrounding structures aiding with surgical planning [61]. Future techniques of printing into deformable biosynthetic materials may facilitate its use in high-fidelity simulators.

Patient-specific simulators have also been developed. A virtual 3D model can be created using a patient's CT data. This allows the surgeon to practice laparoscopic operations in a virtual environment with realistic representations of the patient's anatomy before performing them on the real patient (**Figure 4**) [62]. The simulator's overall accuracy is high, allowing the tumor, arteries, and veins to be visualized. The time it takes to develop these simulations is short, with the hepatectomy and pancreatectomy simulators needing approximately 2.5 hours [63]. As a result, these technologies may have a role in preoperative planning and preparation for complex procedures. However, because patient-specific VR simulators are a novel technology, further research is needed to validate them.

With all models of simulation, an appropriate educational program or curriculum needs to complement its utility. Therefore, research into the applicability of models to facilitate acquiring or mastering procedural skills needs to involve an entire educational program and not just simply time spent on a simulation model. Furthermore, future evidence should focus on measuring and obtaining clinical outcomes associated with high-quality patient care to ultimately demonstrate simulations' effectiveness.


**Author details**

Biswabara Sahu\*, Snigdha Chatterjee and Ruby Patel Department of Agricultural Chemistry and Soil Science, Bidhan Chandra Krishi Vishwavidyalaya, Mohanpur, West Bengal

\*Address all correspondence to<EMAIL_ADDRESS>
© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


**3. Changes concerning cooperative housing**

Until the 1990s, housing cooperatives played a dominant role in meeting housing needs in Poland. They functioned on the basis of the Act of September 16, 1982, Cooperative Law (Journal of Laws No. 30, item 210). It was not until December 15, 2000 that the act on housing cooperatives was passed (Journal of Laws 2001, No. 4, item 27), the aim of which was to create separate legal rules

for the functioning of housing cooperatives, providing cooperatives with greater control of the cooperative's management.

The main goal of the operation of housing cooperatives is to satisfy the housing needs of members of the cooperative and their families. Setting up a cooperative is simple - the minimum requirement is ten founders. A housing cooperative is a legal person, and within it there is a general meeting of members of the cooperative as the highest body, as well as the supervisory board and management board that conducts the day-to-day affairs of the cooperative. The members of the cooperative acting jointly form the general meeting of cooperative members, which must be convened at least once a year. The equality of its members is the principle of the cooperative.

Currently, Polish regulations distinguish two types of rights to cooperative premises that may be granted to members of a cooperative: cooperative tenant rights to a dwelling and cooperative ownership right to a dwelling [10]. Previous regulations also concerned the right to a single-family house in a housing cooperative. The statutes of each cooperative indicate what types of rights the cooperative offers to its members. Each of these rights arises under an agreement concluded by the cooperative with its member. Membership in a housing cooperative is currently granted by law to everyone who is entitled to use the cooperative rights to the premises.

A co-operative tenant right to a dwelling is a right similar to tenancy. A contract specifies the amount of the housing contribution to be made by a member of the cooperative. Only one person may be entitled to it, with the exception of spouses. The cooperative remains the owner of the flat, and the tenant is entitled to use the premises and is obliged to pay the fees. This right is not transferable and not enforceable. In the event of the tenant's death, this right expires, in some cases it is possible for a relative (spouse, children) to enter into the right.

The cooperative ownership right to the premises is included in the category of limited property rights. This right is similar in content to ownership, although the housing cooperative remains the owner of the flat. It may belong to several people. This right is transferable and hereditary, and can also be enforced. A land and mortgage register may be established for such premises, and it may be the subject of collateral in the event of taking out a mortgage. Housing cooperatives are required to keep a register of premises for which a land and mortgage register has been established. Most of the cooperative flats are still operating in the legal form of the cooperative ownership right to the premises, despite the fact that since 1997 its creation has no longer been possible.

The cooperative ownership right to a dwelling was a surrogate for the ownership of a dwelling at a time when the provisions of Polish law did not provide for the legal structure of separate ownership of premises in multi-family buildings. Currently, the legislator considers the cooperative ownership right to the premises to be a redundant form in view of the most powerful form of holding the premises, which is the ownership of the premises. At the same time, the legislator does not interfere with existing rights, and does not force cooperative members to transform them into ownership, but provides such a possibility. Pursuant to the provisions of the Act on Housing Cooperatives, at the request of the entitled person, the housing cooperative is obliged to separate the premises and sell the right to ownership. A member of the cooperative is obliged to pay all fees related to the maintenance of such flat, as well as to reimburse the costs of building the flat, if they have not been covered yet. In addition, the housing cooperative is not allowed to charge any fees for the conversion of rights to the premises. In this way, a housing community may be established in a building previously managed by a housing cooperative, and a departure from the cooperative management regime to the rules of the Act on the

#### *Housing Law in Poland—From the Cooperative Model to Flat Ownership DOI: http://dx.doi.org/10.5772/intechopen.98644*

Ownership of Premises may occur. The trend is noticeable of the loss of management over some buildings by housing cooperatives. This is owing to the separation of the ownership of the premises in them and the decision to entrust management to an entity other than a housing cooperative. Thus, in the resources of housing cooperatives there may be separate premises, which have been created either as a result of the transformation of cooperative rights to premises, or as a result of the construction activity of the housing cooperative after 1997, i.e. when the possibility of creating cooperative ownership rights was abolished.

The number of cooperative flats made available for use in Poland is gradually decreasing. In 2000 there were 24,400 of them, and in 2010 only 5,025 [11]. According to the data as of December 31, 2018, there were 2,030,000 cooperative flats in total (out of a total of 14,615,000 flats in Poland) [12], which means that the number of cooperative flats is still very large. Large housing cooperatives operate in many Polish cities, managing housing estates built in the 1970s and 1980s. Currently, they focus on maintaining the good condition of buildings that require renovation, so their activities are limited to management only. In addition to large cooperatives, there are also small ones that have one or several buildings in their resources. Housing cooperatives in Poland may still carry out construction investments, and the premises may be handed over for use either on the basis of a cooperative tenant's right to a dwelling, or sold as a separate ownership of the premises. Currently, however, the activity of housing cooperatives in the area of erecting residential buildings is small.


**11. Future Considerations**

Impalpable thyroid cancers detected by ultrasound have almost always an excellent prognosis. The precursor lesions of DTC are not well-established and recognised pathology lesions, but there is no clear demarcation that differentiates precancerous from cancerous lesions. If these were to be reclassified as an indolent lesion of epithelial origin (IDLE), the need for aggressive therapy and screening would be mitigated [102]. Therefore, the redefinition of these lesions as "papillary lesions in situ" as precursors of malignant tumours might be beneficial in reducing the overdiagnosis and overtreatment of patients with thyroid nodules [103]. Being able to select which patients would develop more aggressive disease will have huge impact on healthcare costs for DTC.

Surgical complications from an often-unnecessary operation, the emotional distress linked to the diagnosis of 'cancer', and the stress of follow-up, as well as the financial burden to the individual and society, should not be ignored. The problem affects wealthy countries where the steep rise in thyroid ultrasound and FNA has been driven by access to diagnostic imaging. The reversal or slowing down of this trend requires an understanding of the pathology at all medical levels but is not easy to solve [104]. Education whilst helpful clashes with the realities of defensive medicine where the fear of litigation can intimidate doctors towards more investigations, more interventions, and endless follow-up that transforms every person into a lifelong patient. Future research should be directed towards micro-cost analyses to identify potential factors associated with the increased costs. Cost-effectiveness studies with QALY and ICER calculations should be implemented in future guidelines on treatment, surgical, and follow-up strategy.

Some ground-breaking work in health economics has been undertaken, but more needs to be done on to stem the tide and avert medical bankruptcy. Some changes that have been shown to help are the centralisation of cancer care for an economy of scale and quality assurance that comes from group practice and a multidisciplinary environment.

However, a broader, international approach is required to address the problem of overdiagnosis and overtreatment of thyroid cancer, facilitated by data collection, health economic assessment, subspecialisation, and international health policy that together may find a balance between expenses and clinical benefit for the patient. International societies will have to incorporate health economic considerations into their guidelines. The revised 2015 ATA guidelines stated several research questions that remain unanswered to date: optimising molecular markers for diagnosis, AS of DTC primary tumours, and improved risk stratification and survivorship care. Potential answers could all influence future healthcare expenditures.


**Some Collaborative Systems Approaches in Knowledge-Based Environments**

Mihaela I. Muntean

*West University of Timisoara, Faculty of Economics and Business Administration Romania*

#### **1. Introduction**

378 New Research on Knowledge Management Models and Methods

[3] M. E. J. Newman. (2004).*Fast algorithm for detecting community structure in networks*,

[4] M. E. J. Newman. (2005). *A measure of betweenness centrality based on random walks, Social*

[5] H. Small. (1973). *Co-citation in the scientific literature: a new measure on the relationship*

[6] M. Kessler. (1963). *Bibliographic coupling between scientific papers*, American

[7] Lawrence Page, Sergey Brin, Rajeev Motwani, Terry Winograd. (1998). *The PageRank*

[8] M.Tachibori,H.Maruyama,M.Kobayashi,D.Yellin,N.Yoshida,N.Kawai .(2005). *A Survey*

*between two documents*, Journal of the American Society for Information Science,

*Report Digest on Research Trends in Software Architecture*,IPSJ SIG Technical Report,

PHYSICAL REVIEW E 69, 066133, pp1-5, 2004.

Documentation Volume 14, Issue 1, pages 10–25.

*Citation Ranking*. Bringing Order to the Web'.

*Networks*, Vol. 27, No.1, pp. 39-54.

Vol. 24, pp. 28-31.

pp45-52.

Collaboration represents a strategic alternative to the monolithic approach to business development and competition. It involves a different approach to business – focused on managing business relationships between people, within or without groups, and within and between organizations.

Fig. 1. Collaboration – A Business Strategy (http://literacy.kent.edu/CommonGood )

In the present global economy, strongly influenced by IT (information technology) and information systems evolution, the modern organizations try to face the challanges by adjusting their strategies and restructuring their activities, for aligning them to the new economy requirements. It is certain, that the enterprise's performance will depend on the capacity to sustain collaborative work. The evolution of information systems in these collaborative environments led to a sudden necessity to adopt, for maintaining all kind of virtual activities/processes, the latest technologies/systems that are capable to support integrated collaboration in business services.

It is obvious that, all collaborative environments (workgroups, practice communities, collaborative enterprises) are based on knowledge, and between collaboration and knowledge management (KM) there is a strong interdependence. Therefore, we focused on how collaborative systems are capable to sustain knowledge management and their impact on optimizing the KM life cycle. Some issues regarding collaborative systems are explored and a portal-based IT solution that sustains the KM life cycle is highlighted. In addition, an agile development framework for portals will be proposed

Some Collaborative Systems Approaches in Knowledge-Based Environments 381

The orientation toward a collaborative business represents a necessity for developing a competitive business in the actual global economy (Muntean 2006; Ghilic-Micu, Stoica, Mircea, 2008). At the end of 2008, Forrester Research Inc. surveyed business environment inquiring about companies' opening towards adopting new IT collaborative platforms; a trend was obvious, more than 50% heading to collaborative technologies. Based on our research, we propose the adoption of portal platforms at the level of collaborative communities/environments, and followed by integration of a series of systems, collaborative instruments, and enterprise applications which help to consolidate portal's SOA architecture (Service Oriented Architecture) meant to sustain collaboration as a business strategy. It is obvious that collaborative systems maintain knowledge management (KM) at the level of collaborative environments, interfering into all stages of KM life cycle

Without iterating different consecrated approaches in the field of collaborative systems (Mooney, 1974; Giboin, 2000; Marakas, 2003; Crabtree, 2003; Pels, Wortmann, 2003; Cil, Altrup, Yazgan, 2005; Kurdel, Sebestyenova, 2007; Niţchi, 2006, 2007, 2008), we will develop

1. in the actual global economy, in which collaboration is imposed as a business strategy, collaborative environments based on any kind of knowledge presume the existence of informatics systems, tools and technologies being able to maintain collaborative work, collaborative solving of different tasks, collaborative decisions and consolidating value; 2. multi-agent systems imposed themselves as facilitating collaboration, maintaining economic processes/activities, as well as collaborative solving of specific complex

3. on the other hand, collaborative tools are indispensable in every collaborative organization/community, concurring on sustaining the knowledge dissemination

4. but, the IT infrastructure of collaborative communities presumes the existence of complex platforms, with various capabilities in order to virtualize a big part of the

This can be achieved only with the help of portal technologies, at their level being integrated multi-agent systems with specific functions as well as a diverse range of conversational

Multi-agent systems (MAS) are computer based environments that contain multiple software agents to perform certain tasks (Turban, 2007). Multi-agent systems offer an innovative perspective on the capacity of configuration virtual communities. But some of

portal-based IT platforms; initiative sustained by the following considerations:

(subject will be developed in paragraph 4).

problems;

tools.

between its members;

communities' reality.

our scientific intercession around some consideration towards:

 collaborative systems based on software agents; collaborative tools (in fact, conversational tools);

**3.1 Considerations regarding multi-agent systems**

aiming autonomy, cooperation and learning;

languages (KQML, KIF, COOL, etc.);

their capabilities must be perfected, as follows (Muntean, 2009):


According to the changes of relative abundance and trophic structure of protists by the rank sum test of Kruskal-wallis and Nemenyi-Wilcoxon-Wilcox, it showed that P promoted the growth of protists with the different particle sizes and the trophic modes at the early stage of culture. While the sand-dust presented different effects on the growth of different trophic modes. It stimulated the autotrophic but inhibited the heterotrophic and mixotrophic protists. The order of relative abundance of autotrophic protists with different particle sizes was 10–20 μm > 5–10 μm > 2–5 μm. The order of decreasing the proportion of heterotrophic protists with different particle sizes was 5–10 μm > 10–20 μm > 2–5 μm; and the order of inhibiting the proportion for mixotrophic protists with different particle sizes was 10–20 μm > 5 μm > 2–5 μm. The early stage of sand-dust deposition obviously inhibited the heterotrophic and mixotrophic protists by the dissolution of toxic and harmful substances in sand-dust. Mixotrophic and heterotrophic protists are the primary predator of autotrophic protists; Pearce et al. (2011) found 42%~82% primary production was consumed by mixotrophic and heterotrophic protists [35]. The decrease of mixotrophic and heterotrophic protists could accelerate the growth of autotrophic protists. In this study, sand-dust addition promoted the growth of autotrophic protists at the early stage, and the decrease of mixotrophic and heterotrophic protists accelerated this process. The further study confirmed that sand-dust storm could significantly induce the occurrence of red tide in the southern Yellow Sea [18,30]. Besides, the harmful inhibiting effect of sand-dust on heterotrophic protists community would weaken the marine matter cycle and food transfer efficiency.

At the late stage of culture, P presented a prohibitive effect on the growth of protists, which was related to the rapid growth of protists at the earlier stage, which resulted in nutritional deficiency and growth restriction at the late stage. In the late stage of sand-dust deposition, the autotrophic protists were limited due to the excessive growth of autotrophic protists in the earlier stage. At the late stage, sand-dust demonstrated stimulating effect for heterotrophic and mixotrophic protists, and the order was heterotrophic > mixotrophic protists. As for the heterotrophic protists with different particle sizes, the order was 5–10 μm > 2–5 μm > 10–20 μm, and for mixotrophic protists, the order was 10–20 μm > 5–10 μm > 2–5 μm. In the late stage of sand-dust deposition, the toxicity of harmful substances to heterotrophic protists gradually weakened. These protists slowly adapted to the sand-dust environment and dissolution of organic substances and other nutrients in sand-dust provided abundant nutrients for the heterotrophic protists [17]. As such, their abundance maintained stable and their proportion gradually increased. The effects of low sand-dust group and high sand-dust group on the proportion of heterotrophic and mixotrophic protists were basically coincident. However, the effect of high sand-dust group was stronger and the nutritional supplementation lasted longer in the later period.

For the trophic structure, at the early stage of incubation, P addition could promote all kinds of phytoplankton protists, especially the 10–20 μm autotrophic protists, which were consistent with that of autotrophic protists at the early stage of sand-dust deposition. At the late stage of incubation, both P addition and sand-dust deposition restricted all sizes of autotrophic protists. Therefore, it is speculated that the effect of sand-dust deposition on the autotrophic protists in this ocean were ascribed to the dissolution of P from sand-dust. The early promotion of sand-dust was to supplement the P in the sea area, while at the late stage, the inhibition for the autotrophic protists was mainly related to the early rapid growth of autotrophic protists by consuming up the P and other nutrients. The limiting capacity of the sand-dust group at the late stage is less than that at P-adding groups. This reflected that the dust could continuously supply the P and other nutrients.

The abundant P at the early stage of sand-dust deposition promoted the rapid growth of large-size autotrophic protists. This phenomenon will further accelerate the occurrence of red tides in eutrophic sea areas. Sand-dust deposition in the late stage stimulated the small-size heterotrophic protists and accelerated the material cycle efficiency and food transfer capacity in the sea. Therefore, the influence of sand-dust on the structure of different particle size and trophic protists will change the structure of the micro-food web in the ocean. It will also change ability of material transformation in the water body and ultimately, and affect the ecological function of protists in the transforming matter and producing food in the sea.

#### **5. Conclusions**

(1) Sand-dust deposition affected the trophic structure of different particle sizes of planktonic protists in the southern Yellow Sea. This could lead to change the structure of micro-food webs in the sea, and affect the ecological functions of micro-food webs in material transformation and food production.

(2) The growth of planktonic protists of all trophic modes in this ocean was restricted by P. The early addition of P could promote the growth of planktonic protists of all trophic modes in the southern Yellow Sea. The effect on the10–20 μm autotrophic protists was most obviously, while the late addition of P mainly restricted the growth of different sizes of protists.

(3) The effect of initial sand-dust deposition on autotrophic protists was the same as that of P, it inhibited heterotrophic and mixotrophic protists. The positive effect of sand-dust deposition on heterotrophic and mixotrophic protists was strong at the late stage, and it improved the abundance of small-sized heterotrophic protists.

(4) The positive effect of sand-dust deposition on autotrophic protists in the Yellow Sea might be related to the dissolution of P from the sand-dust. The promotion of small-size heterotrophic protists in the late stage of sand-dust deposition could accelerate the material circulation efficiency and food transformation in the sea.

**Author Contributions:** Conceptualization, X.C. and Y.-G.Z.; methodology, X.C., G.-X.L. and H.-J.C.; investigation, X.C., H.-J.C., and C.Z.; formal analysis, X.C. and C.Z.; project administration; G.-X.L. and H.-J.C.; resources, G.-X.L. and H.-J.C.; software, X.C.; supervision, G.-X.L.; writing—original draft preparation, X.C., and Y.-G.Z.; writing—review and editing, X.C., Y.-G.Z. and X.H.; funding acquisition, G.-X.L.; validation, G.-X.L.; visualization, X.C and X.H.

**Funding:** Financial support for this project was provided by the National Natural Science Foundation of China (NSFC; 41210008) and the Major State Basic Research Development Program of China (973 Program; 2014CB953701). This study was conducted at the Ocean University of China.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Stable Isotope Analysis and Persistent Organic Pollutants in Crustacean Zooplankton: The Role of Size and Seasonality**

#### **Roberta Piscia 1,\*, Michela Mazzoni 1,2, Roberta Bettinetti 1,2, Rossana Caroni 1, Davide Cicala 1 and Marina Manca 1**

Received: 12 June 2019; Accepted: 14 July 2019; Published: 18 July 2019

**Abstract:** Zooplankton is crucial for the transfer of matter, energy, and pollutants through aquatic food webs. Primary and secondary consumers contribute to the abundance and standing stock biomass, which both vary seasonally. By means of taxa- and size-specific carbon and nitrogen stable isotope analysis, the path of pollutants through zooplankton is traced and seasonal changes are addressed, in an effort to understand pollutant dynamics in the pelagic food web. We analyzed zooplankton plurennial changes in concentration of polychlorinated biphenyls (PCBs) and dichlorodiphenyltrichloroethane and its relatives (DDTs) and in taxa-specific δ15N signatures in two size fractions, ≥450 μm and ≥850 μm, representative of the major part of zooplankton standing stock biomass and of the fraction to which fish predation is mainly directed, respectively. Our work is aimed at verifying: (1) A link between nitrogen isotopic signatures and pollutant concentrations; (2) the predominance of size versus seasonality for concentration of pollutants; and (3) the contribution of secondary versus primary consumers to carbon and nitrogen isotopic signatures. We found a prevalence of seasonality versus size in pollutant concentrations and isotopic signatures. The taxa-specific δ15N results correlated to pollutant concentrations, by means of taxa contribution to standing stock biomass and δ15N isotopic signatures. This is a step forward to understanding the taxa-specific role in pollutant transfer to planktivores and of zooplankton enrichment in PCBs and DDTs.

**Keywords:** stable isotope analysis; persistent organic pollutants; crustacean zooplankton; freshwater; size fractions; seasonality

#### **1. Introduction**

In lakes, crustacean zooplankton are low-order consumers and represent an important link between the base of the pelagic food web and the organisms at higher trophic levels, which may have economic or conservation value. Although considered a homogeneous compartment, zooplankton is composed of organisms that differ substantially from each other not only in their taxonomy, but also in body size, metabolic rate, and ecological roles [1]. While heterogeneity of the zooplankton community has been fairly well-documented in basic ecological studies, it is often overlooked in ecotoxicological ones, particularly in models, which are usually focused mainly on sources and top-levels of pollution patterns, the latter being directly related to human health. In particular, it is a crucial step in the flow of pollutants through aquatic ecosystems [2–5]. Both these pathways are involved, because zooplankton is directly related to primary producers and is able to process organic matter to feed on phytoplankton and to actively contribute to the microbial loop [6,7]. Zooplankton also represents a food source for all fish types at the larval stage and for zooplanktivorous fish at the adult stage [8,9]. Predation by fish and vertebrates is, in general, visual, therefore depending on prey size and visibility [10]. Both primary and secondary consumers may contribute to standing stock biomass in each size fraction considered. The contribution of primary and secondary consumers to zooplankton standing stock biomass generally varies with the season in deep, temperate, subalpine lakes, such as Lake Maggiore. In deep, temperate lakes, earlier spring warming and conditions of thermal water stratification influenced by climate change promote an early spring peak in phytoplankton biomass, followed by a peak in zooplankton biomass, mainly due to primary consumers, after about one month. The subsequent slow decline of zooplankton primary consumers is concomitant with an increasing contribution of zooplankton secondary consumers to the total zooplankton biomass in late summer. This pattern of change in zooplankton community composition also includes a change in organism size; in fact, a prevalence of larger sizes is observed when the contribution of predatory zooplankters increases.

Lake Maggiore has a long history of severe pollution brought about by persistent organic pollutants, such as dichlorodiphenyltrichloroethane and its metabolites. The International Commission for the Protection of the Italian–Swiss Common Waters (CIPAIS) supports monitoring activities in this lake, with a particular focus on priority substances, such as DDx and other organic chemicals of industrial origin, such as polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs), known for their harmful effects on human health and on environment [11,12]. Changes in physical and chemical conditions may cause their partial release into the dissolved phase, with increased bioavailability for organisms [13]. Because of their physico-chemical properties, persistent organic pollutants (POPs) can be detected everywhere, including worldwide remote regions far away from emission sources, such as polar or Alpine ecosystems [14–17]. In this study, we analyzed the seasonality of zooplankton in relation to persistent organic pollutant concentrations (i.e., DDTs and PCBs) in two different size fractions, one representative of most zooplanktonic biomass (≥450 μm) and the other representative of fish food source (≥850 μm). The former is representative of the major part of the crustacean zooplankton, while the latter represents the size fraction to which fish predation is mainly directed [10,18]. Despite its importance, zooplankton body size is usually neglected in studies on trophic pollutant transfer. Both seasonality and size are expected to drive zooplankton pollutant concentration over the season and a proper biomass estimation is essential to determine whether there is a prevalence of either of the two drivers. Furthermore, in order to study the modifications in the food web, we used carbon and nitrogen stable isotope analysis (13C, 15N; SIA). By this method, food sources (e.g., littoral versus pelagic, deep versus shallow) can be clearly identified in a system, as can the time-specific contribution of organisms to food webs. The method was originally applied to marine environments and to fish in particular, not least for fingerprinting their origin. Only recently it has been increasingly applied in freshwater environments, not only regarding fish or other organisms of direct human consumption, but also regarding those which sustain their production and growth, thus allowing for a more reliable reconstruction of the food web [19]. SIA is applied to investigate ecosystems in the making, but also to predict, by means of organism nitrogen isotopic signature, timeand space-specific trophic position, from which, among others, biomagnification depends [20–22]. The trophic role and food sources of organisms can be evaluated by carbon and nitrogen stable isotope analysis [23–25]. Isotopic signatures provide information on the resources exploited by a consumer within an environment and its trophic positions in the food web [24,26]. In particular, the isotopic carbon signature is almost constant between food source and consumer, whereas a nitrogen isotopic enrichment is observed with an increase of the trophic status [27]. In the present work, we related the trophic role of zooplankton with persistent organic pollutant (POPs) concentrations in Lake Maggiore (northern Italy). Previous long term studies in this deep, large Italian lake suggested that zooplankton seasonal dynamics and the different primary and secondary consumer contributions to zooplankton biomass are reflected by different contributions of the two important native predatory cladocerans (*Bythotrephes* and *Leptodora*), of diaptomids and cyclopoid copepods, and of the cladoceran *Daphnia*.

Our purpose is to investigate, in particular, how DDTs and PCBs vary with the season and in the two different size fractions, the former representing the bulk of crustacean zooplankton biomass and the latter representing the size fraction on which fish predation is mainly oriented. We hypothesize that variations are related to changes in δ15N signature of the two fractions and that the latter results from the taxa-specific contribution to zooplankton biomass. We expect to find a correlation between taxa-specific signatures and taxa contribution to measured zooplankton standing stock biomass.

#### **2. Study Site**

Lake Maggiore is the second deepest (Depthmax = 370 m) and largest (area = 212 km2, volume 37.5 km3) subalpine lake located in the northwestern part of Italy (Figure 1). By its nature oligotrophic, the lake underwent eutrophication during the 1960s and 1970s. The eutrophication reversal and the return to oligotrophic conditions were due to a reduction in total phosphorus discharge into the lake after wastewater treatment facilities were opened and thanks to the ban of phosphorus-containing detergents.

**Figure 1.** Map of Lake Maggiore. Sampling station location is shown with the red dot (from Google Earth).

Since 1996, contamination by dichlorodiphenyltrichloroethane and its relatives (DDTs), produced by a farm plant located in the lake catchment basin was detected [28]. Polychlorinated biphenyls (PCBs) in a steady-state condition were also reported [29–32].

Lake Maggiore is one of the best-studied lakes in Italy, with the first ecological research, including studies on zooplankton population dynamics, dating back to the early 1900s [33,34]. Long-term studies on zooplankton seasonal dynamics have been conducted over the last ten years by C and N stable isotope analyses, aimed at identifying trophic interactions and taxa-specific roles in the transfer of matter and pollutants within the pelagic food web.

#### **3. Materials and Methods**

Zooplankton sampling was performed seasonally at Ghiffa station (45◦57' N; 8◦38 E; Figure 1) in correspondence with the lake's deepest area (370 m). We collected two samples every day using two zooplankton nets that were 58 cm in diameter (450 μm and 850 μm in mesh size), which were hauled from 0 to 50 m deep several times in order to obtain a sufficient amount of organisms needed to perform the analyses (min = 2, max = 15). The total volume filtered was approximately 26 m3 per zooplankton collection. Samples of the size fraction ≥450 μm were collected from January 2012 to January 2016, while samplings of the size fraction ≥850 μm started later (May 2013). All samples were collected in duplicate. Live organisms of one of the two samples were concentrated in a laboratory setting in approximately 1 L of lake water, frozen at −20 ◦C, and subsequently used for carbon (13C) and nitrogen (15N) stable isotope analysis (SIA). One third of the second zooplankton sample was immediately fixed and preserved in 95% ethanol for microscopic analysis at 6.3×. We calculated the biomass of the different taxa by way of length–weight regression equations [35,36]. On the basis of the taxa, zooplankton organisms were grouped into primary consumers (herbivores, i.e., *Daphnia*, *Eubosmina*, *Diaphanosoma,* and copepod diaptomids) and secondary consumers (predators, i.e., *Bythotrephes*, *Leptodora,* and copepod cyclopoids). The remaining portion of the second sample (two-thirds) was filtered on a 1.2 μm pore glass–fiber-filter (GF/C, 4.7 cm of diameter) and then frozen at −20 ◦C. As a consequence of using large net mesh sizes, rotifers and early developmental stages of crustacean zooplankton (i.e., the smallest body size organisms) and large phytoplankton colonies were not included in our samples. However, they are of only marginal importance for the purpose of our study, in which large zooplankton was regarded as an important link in the transfer of pollutants from primary producers to fish owing to selective predation on visible prey, particularly large-bodied zooplankton [37]. SIA was performed on pooled samples of the two size fractions (≥450 μm and ≥850 μm) and on the already-listed crustacean taxa present in sufficient amounts in the sample. Under the dissecting microscope, we pooled individuals of each taxon in order to reach the minimum dry weight (DW) of 2 mg per sample (approximately 70–700 individuals depending on the biomass). Samples were oven-dried for 24 h at 60 ◦C, before being homogenized and transferred into tin capsules of 5 × 9 mm in size. The isotopic compositions of organic carbon and nitrogen were determined by Ján Veizer Stable Isotope Laboratory (Ottawa University, Ontario, Canada) following methods already described in Visconti and Manca [38]. Isotope ratios were expressed as parts per thousand (%) difference from a standard reference of PeeDee Belemnite for carbon and atmospheric N2 for nitrogen, according to the equation:

$$^{13}\text{C}\_{\prime}\text{ }^{15}\text{N}=\left[\left(\frac{\text{R}\_{\text{sample}}}{\text{R}\_{\text{standard}}}\right)-1\right]\times 1000\tag{1}$$

where R is the isotopic ratio 13C/ 12C and 15N/ 14N.

For determination of organic compounds (OCs), the materials and methods described in Mazzoni et al. [1] were followed. Quantitative DDT homologue analyses were performed by the external standard method using a solution containing pp'DDT, pp'DDE, and pp'DDD as the reference standard, prepared from single pure compounds (Pestanal, Sigma–Aldrich, Steinheim, Germany) in iso-octane (Carlo Erba, pesticide analysis grade, Val de Reuil, France). Arochlor 1260 (Alltech, IL, USA), while the addition of PCB 28, 52, and 118, was used for PCB quantification. The analyzed congeners consisted of PCB 28, 52, 101, 118, 138, 149, 153, 170, and 180. The detection limit for each OC was 1 ng·g−1 lipid weight (l.w.).

We used the software SigmaPlot 11.0 (version 11, Systat Software Inc., San Jose, CA, USA) to perform statistical analyses. After verifying that the datasets were normally distributed, we applied two-way ANOVA in order to identify which factor, size or seasonality, drove changes throughout the year.

#### **4. Results**

A strong seasonality characterized stable isotope data and zooplankton biomass composition. In Figures 2 and 3, we reported data of taxa/groups biomass percentage composition, δ13C% and δ15N% values and total crustacean biomass of the two size fractions analyzed (≥450 μm and ≥850 μm). A remarkably higher isotopic carbon (or less depleted) signature in summer and a higher nitrogen signature in autumn and winter was observed in the samples of both size fractions. The most depleted carbon signatures in the fraction ≥850 μm occurred with high relative abundance of primary consumers

to total zooplankton biomass, concomitant to the spring peak or abundance of the cladoceran filter feeder *Daphnia*. In the ≥450 μm fraction, where most depleted carbon signatures were recorded, cyclopoids and diaptomids were also important contributors to the total biomass, together with Cladocera filter feeders.

**Figure 2.** (**A**) Biomass percentage composition of planktonic crustacean taxa (bubbles) of Lake Maggiore and their δ13C% values (bubble centers) and δ13C% values of pooled samples (black line); (**B**) biomass percentage composition of planktonic crustacean taxa (bubbles) of Lake Maggiore and their δ15N% values (bubble centers) and δ15N% values of pooled samples (black line); (**C**) total biomass of Lake Maggiore planktonic crustacean taxa. All data refer to the fraction ≥450 μm.

Zooplankton standing stock biomass largely changed with the seasons. In samples belonging to the ≥450 μm size fraction, a spring peak was followed by a second phase of biomass increase in autumn.

In the ≥850 μm size fraction, peak biomass values were generally recorded in August. The unusual increase in May 2015 was related to the detection of the peak population growth of *Daphnia*. As already highlighted, *Daphnia* can be an important contributor to standing stock biomass, due to its peak biomass in spring being related to the development of young, smaller specimens.

We observed a clear seasonality of δ13C isotopic signatures for the ≥450 μm size fraction during the studied years. The least depleted values in δ13C% were regularly measured in August, while more depleted values characterized the spring samples (May), when the contribution to the peak biomass of primary consumers, particularly *Daphnia*, was at its peak. Limited seasonal variation regarding the contribution to the total biomass was characteristic of the cyclopoid copepods, whose contribution to carbon and nitrogen isotopic signatures showed similar values during the year. In the >450 μm size fraction, cyclopoids and calanoids were related to depleted values of carbon isotope signatures and to the highest values of nitrogen signatures. The biomass contributions of different zooplankton taxa, both primary and secondary consumers, drove patterns of both isotopic signatures; therefore, the measured values, e.g., in January 2012, integrated isotopic signatures of the two taxa present, i.e., cyclopoid copepods and, to a lesser extent, *Daphnia*.

**Figure 3.** (**A**) Biomass percentage composition of planktonic crustacean taxa (bubbles) of Lake Maggiore and their δ13C% values (bubble centers) and δ13C% values of pooled samples (black line); (**B**) biomass percentage composition of planktonic crustacean taxa (bubbles) of Lake Maggiore and their δ15N% values (bubble centers) and δ15N% values of pooled samples (black line); (**C**) total biomass of Lake Maggiore planktonic crustacean taxa. All data refer to the fraction ≥850 μm.

A marked seasonality was also characteristic of the δ13C and δ15N signatures in the ≥850 μm size fraction. The least carbon-depleted values in August were related to the high contribution of secondary consumers, while more carbon-depleted values in spring and winter were related to a higher contribution of primary consumers. Values tended to be most enriched in winter, with secondary consumers more enriched than primary ones. The signatures of δ15N% measured in the ≥850 μm size fraction were related to the relative biomass contribution of primary and secondary consumers. Among predatory cladocerans, *Bythotrephes* was generally much more δ15N enriched than *Leptodora*. For example, in August 2013, the nitrogen enrichment of *Bythotrephes* was higher in comparison to *Leptodora* and *Daphnia*. Differences in δ13C signatures suggest that *Leptodora* was living in deeper waters than *Bythotrephes* and *Daphnia*. Changes in isotopic signature and contribution to total biomass were mainly related to cladoceran primary and secondary consumers, thus enabling the investigation of infra-Cladocera food relationships.

As hypothesized, we found that carbon and nitrogen isotopic signatures of the two pooled size fractions could be reconstructed from taxa-specific signatures weighted on contribution to total biomass, by applying the following equation:

$$\delta^{15} \text{N}\_{\text{\textquotedblleft}} \delta^{13} \text{C}^{\text{\textquotedblleft}} \text{\textquotedblright} \text{\textquotedblright} \text{\textquotedblright} \text{\textquotedblleft} = \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblright} \text{\textquotedblleft} \text{\textquotedblright} \text{\textquotedblright} \text{\textquotedblright} + ... + \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblright} \text{\textquotedblright} \text{\textquotedblright} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblright} \text{\textquotedblright} \text{\textquotedblright} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedblleft} \text{\textquotedbl$$

where *i* = δ15N% and δ13C% are taxa-specific signatures.

Relationships between measured and estimated isotopic signatures were significant (δ13C%: R2 = 0.904, N = 28, F = 244.682, *P* < 0.001; δ15N%: R2 = 0.942, N = 28, F = 438.413, *P* < 0.001; Figure 4). Such relationships are the result of the accuracy of estimation of both biomass and taxa-specific isotopic signatures. In fact, taxa-specific biomass estimation is based on length–weight regression equations applied to a consistent number of specimens (at least 25 specimens per taxa) and taxa-specific isotopic signatures analyses are based on a very high number of specimens (cfr. Section 3), which is necessary

for Isotope Ratio Mass Spectrometry (IRMS) analysis (at least 1 mg of dry weight). The accuracy of standing stock biomass determination from length–weight regression of taxa-specific analysis was also confirmed by the comparison with the direct biomass dry weight estimation, which was shown to be statistically significant (R2 = 0.814, N = 8, F = 26.251 *P* = 0.002).

**Figure 4.** Relationship between isotopic fingerprint (carbon and nitrogen) of pooled zooplankton samples belonging to ≥450 μm and ≥850 μm size fractions determined by instrumental analysis (measured) and reconstructed values (estimated) following Equation (2).

The difference between the smaller and the larger size fraction was mainly determined by cladoceran taxa composition. In the case of DDTs, the main effects cannot be properly interpreted because a weak, but significant, interaction between size and seasonality was determined when two-way ANOVA was applied (N = 28, F = 3.149, *P* < 0.048), so that the size of a factor's effect depends upon the level of the other factor. Changes in PCBs and δ15N values were shown to be strongly driven by seasonality (PCB: N = 28, F = 14.312, *P* < 0.001; δ15N: N = 28, F = 29.169, *P* < 0.001) and for both datasets there was no statistically significant interaction between factor size and seasonality (*P* = 0.181 and *P* = 0.596, respectively). Results of the two-way ANOVA are reported in Table 1.

A regression analysis was performed with δ15N data and pollutant concentrations (Figure 5) with ≥450 μm and ≥850 μm size fractions. Changes in both DDT and PCB concentrations on a logarithmic scale were related to zooplankton δ15N% isotopic signatures (DDTs: R2 = 0.541, N = 26, F = 28,340, *P* < 0.001; PCBs: R2 = 0.502, N = 27, F = 25.159, *P* < 0.001). The data in the upper right side of the graph

refer to autumn and winter samples, while those in the lower side of the graph are related to spring and summer samples.

**Table 1.** Results of the two-way ANOVA tests performed on DDTsPCBs concentrations and δ15N% fingerprint of zooplankton samples of the two size fractions (≥450 μm and ≥850 μm).

**Figure 5.** Relationship between nitrogen isotopic fingerprint of pooled zooplankton samples from Lake Maggiore, belonging to ≥450 μm and ≥850 μm size fractions, and persistent organic pollutant (POPs) concentrations, expressed as logarithmic scale.

POP concentrations in the ≥850 μm size fraction were generally lower than in the ≥450 μm size fraction (Figure A1).

#### **5. Discussion**

The pelagic zooplankton is composed of a large variety of organisms differing in taxonomic composition and body size. Together, protists, monogonont rotifers, Cladocera, and Copepoda contribute to the zooplankton community, the latter with both individuals at their adult stage and a number of developmental stages that are different in size and trophic role. Regarding net zooplankton, as a result of the mesh size, only rotifers and crustaceans were reliably sampled. The former can be numerically dominant during the spring phase, along with naupliar and early copepodite stages of copepods. Their contribution to zooplankton biomass is little, however, with respect to Cladocera and adult or sub-adult copepods [38–40]. Altogether, the latter are ≥450 μm in size.

Zooplanktivorous fish, however, tend to select larger (≥850 μm) and more visible prey, such as *Bythotrephes*, large, ovigerous *Daphnia*, and *Leptodora*. Therefore, these are a component of the zooplankton population, which is active in transferring POPs to fish [10,41–44].

By choosing to focus on the two size fractions of crustacean zooplankton, we aimed to investigate components which are directly involved in the transfer of pollutants in the trophic chain. In addition, investigating the role of micro-zooplankton is almost impossible, given that POP concentrations and δ13C and δ15N analyses require high weight amounts of zooplankton material (approximately 1 μg dry weight and 1 mg dry weight, respectively). Further studies are required to elucidate the role of micro-zooplankton as carrier of pollutants.

As expected, zooplankton biomass composition largely varied with the season in both size fractions analyzed, because in deep subalpine lakes, the abundance and composition of zooplankton populations vary along the season [45–47]. A spring peak abundance of phytoplankton population triggers the growth of primary zooplankton consumers, such as the large filter-feeder *Daphnia*, which, in Lake Maggiore, usually reaches its peak population density in May [47]. The increase of *Daphnia* and other primary consumer cladocerans (*Eubosmina*, *Diaphanosoma*) in spring is followed by an increase in predatory cladocerans (*Bythotrephes* and *Leptodora*), able to selectively exploit primary consumers and contribute to their decline [48,49]. In turn, the decrease in zooplankton primary consumers promotes a second phytoplankton peak in summer, which leads to a second phase of increase in primary consumers in autumn.

In this study, seasonality was also observed in isotopic carbon and nitrogen zooplankton signatures. In pelagic zooplankton, δ13C% tended toward less depleted values in summer, likely mirroring phytoplankton isotopic signatures [38,39,50]. During thermal stratification, the high growth rates of phytoplankton cells can lead to the consumption of dissolved atmospheric CO2, causing a shift in carbon exploitation sources toward the uptake of the HCO3 − ion [51–54]. The unselective filter feeder *Daphnia* feeds on seston particles, which fit the distance between their intersetular filtering combs (i.e., 0.2–50 μm) [55]. Since Lake Maggiore seston is mainly composed of phytoplankton cells and carbon fractionation is negligible, *Daphnia* isotopic signature specimens matched variations of the phytoplankton carbon fingerprint [45,50]. The pelagic zooplankton nitrogen isotopic signature also varied with the season, with the maximum enrichment in winter. Isotopic nitrogen tended to increase along the trophic food chain and stepwise enrichment varied in different environments and with the structure of the trophic web [45,56,57]. Isotopic nitrogen enrichment is diet-dependent [58]. In eutrophic lakes, where high values of δ15N% are usually found, zooplankton organisms do not feed only on phytoplankton, but also exploit alternative organic particulate matter, such as bacteria, protozoa, exuviae, and fecal pellets of zooplankton specimens, which are more δ15N-enriched than phytoplankton algae. We can hypothesize that, in winter, when the abundance of phytoplankton cells is low, alternative, δ15N-enriched food sources (e.g., bacteria, protozoa, exuviae) became more important, leading to the observed enrichment in isotopic nitrogen content. In addition, under food shortage conditions, the zooplankton might be able to exploit δ15N%-enriched lipids which accumulate in high food concentration conditions [59,60].

Distinguishing between primary and secondary consumers is crucial for the reconstruction of patterns of matter and energy through the pelagic food web. As primary and secondary consumers largely overlap each other in body size, such distinction cannot be based on size fraction analyses of zooplankton. We found that the isotopic signature of pooled zooplankton samples of the two size analyzed fractions was tightly related to taxa-specific contribution to total biomass, thus allowing for the identification of taxa-specific contribution to measured δ15N% isotopic signatures. The latter is, in turn, correlated to POP concentrations, therefore allowing for a better definition of primary and secondary zooplankton consumer taxa in the transfer of toxicants through the pelagic food web. The significant relationship between pooled zooplankton samples and taxa-specific contributions to the total biomass in Lake Maggiore results from the fact that other zooplankton particles are excluded when samples are filtered through 450 μm nets. In Lake Maggiore, detritus is usually <126 μm and the contribution of phytoplankton colonies >76 μm is largely negligible. Therefore, any other zooplankton contribution to the total biomass in the samples of the two size fractions analyzed was certainly negligible. This is not the case in eutrophic or mesotrophic lakes, where zooplankton is smaller and detritus and phytoplankton colonies can be found in zooplankton samples in non-negligible amounts [1,61].

We hypothesized that both size and seasonality can influence POP concentrations in zooplankton populations and that the two parameters are related to the δ15N% isotopic signature. Our results highlighted a predominance of seasonality versus size. The ≥450 μm fraction was composed of primary (*Daphnia*, *Eubosmina*, *Diaphanosoma* and diaptomids) and secondary consumers (cyclopoids, *Bythotrephes* and *Leptodora*) throughout the year, while in the ≥850 μm fraction, the biomass of secondary consumers is dominant in August. As secondary consumers are expected to be higher in POP concentrations, we would have expected to record the highest values of POP concentrations in summer; on the contrary, we observed high POP concentrations in winter, when zooplankton nitrogen enrichment was at its maximum. In fact, the δ15N% and POP results directly correlated, with both tending to increase from spring to winter. Ecotoxicological studies on marine trophic chains demonstrated a similar relationship along the food chain [62,63]. In Lake Maggiore, the seasonal pattern of POP concentrations was very similar terms of values between years of the study period [28], suggesting a steady-state condition. In these conditions, the major δ15N-enrichment of carnivorous species was probably hidden by seasonal variations in δ15N%, which was dependent on quality of organic particles, i.e., the bacteria, protozoa, or organic particles deriving from dead organisms should have higher POP concentrations than phytoplankton cells.

The predominance of seasonality versus size was confirmed also by DDT concentrations in the smallest size fraction. Theoretically, the big predators, *Bythotrephes* and *Leptodora*, and the largest *Daphnia* specimens of the ≥850 μm size fraction should be characterized by higher values of POP concentrations, the former due to this specimen belonging to an upper trophic level and the latter because adults of a larger size can accumulate more pollutants in their tissues. However, our results likely reflect the fact that copepods rich in lipids (substance where POPs are mainly stored) were not present in the larger size fraction. However, because zooplanktivorous fish mainly select large cladocerans [43], the ≥850 μm fraction should be more representative of fish food sources.

#### **6. Conclusions**

Zooplankton play a crucial activity in matter and energy transfer in the food web. In temperate lakes, biomass, taxa, and size composition largely vary with the season. The contribution of primary and secondary zooplankton consumers also varies seasonally, along with the δ15N% isotopic signature. By investigating seasonal changes in biomass and taxa-specific contributions and in carbon and nitrogen isotopic signatures along with DDT and PCB concentrations, we aimed at quantifying the relative importance of size versus seasonality. We found that the δ15N% of two size fractions, representative of bulk crustacean zooplankton biomass and of the fraction on which fish mainly prey, respectively, significantly correlated to POP concentration, as both varied with the season. The bulk zooplankton nitrogen isotopic signature resulted from a biomass-weighted taxa-specific isotopic signature, thus enabling us to distinguish between the contributions of secondary and primary

consumers to measured nitrogen enrichment, from which the concentration of pollutants depends. DDT concentration was higher in the >450 μm fraction, while no difference was found in PCB concentration in the two size fractions. The difference between the two was mainly related to copepod adults, which were entirely lacking in the larger, >850 μm size fraction, the one on which fish selectively prey. Overall, seasonality was largely predominant over size for the dynamics of DDTs and PCBs, therefore suggesting that, in temperate lakes, more than one time spot studies are required; indeed, multi-year studies are ideal for verifying, among others, the condition of a steady state which must be fulfilled before applying models regarding the transfer of pollutants through an ecosystem.

The oligotrophic, subalpine Lake Maggiore was ideal for investigating the role of zooplankton in pollutant transfer, as size spectra distinction allows for the neglect of background noise caused by micro-zooplankton, protists, and phytoplankton algae. This is not the case for more productive lakes, in which such size-based distinction cannot be applied in the same way.

**Author Contributions:** Formal analysis, M.M.M. and R.P.; SIA and zooplankton investigation, M.M.M. and R.P.; POPs investigations, M.M. and R.B.; writing—original draft preparation, M.M.M., R.P., R.C., and D.C.; Review, R.B. and M.M.

**Funding:** This research was funded by CIPAIS (Commissione Internazionale per la Protezione delle Acque Italo-Svizzere).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Appendix A**

**Figure A1.** Concentration of POPs in two crustacean zooplankton size fractions (≥450 μm and ≥850 μm) of Lake Maggiore.

#### **References**

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