**C/Li2MnSiO4 Nanocomposite Cathode Material for Li-Ion Batteries**

Marcin Molenda, Michał Świętosławski and Roman Dziembaj

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/48319

## **1. Introduction**

60 Composites and Their Properties

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Technological development of portable devices, e.g, mobile phones, laptops, etc., as well as progress in electrical vehicles (EV) and hybrid electrical vehicles (HEV) technologies require batteries efficient in volumetric and gravimetric energy storage, exhibiting large number of charge/discharge cycles and being cheap and safe for users. Moreover, materials used in energy storage and conversion systems should be environmentally friendly and recyclable. Currently, rechargeable lithium-ion batteries (LIBs) are the most popular portable energy storage system, mostly due to their highest energy density among all others rechargeable battery technologies, like Ni-Cd or Ni-MH cells which reached the theoretical limit of performance. Commercially available LIBs are based on layered lithium cobalt oxide (LiCoO2) or related systems, which are expensive and toxic. These materials are unstable in an overcharged state, thus the battery safety is affected, especially in high power (20-100 kWh) applications for EV, HEV and renewable energy systems. The bigger battery capacity results in more energy accumulated, thus operational safety is a key issue. On the other hand, lifetime and capacity retention of LIBs in changeable operation conditions (from -30°C to +60°C, average lifetime 2-4 years) are a challenges to develop new materials and cell assembly technologies.

## **2. Li-ion battery technology**

First rechargeable lithium cells taking advantages of intercalation process were developed in year 1972 [1]. The cells Li/Li+/LixTiS2 revealed 2V potential and relatively low gravimetric capacity. Applications of metallic lithium as anode material resulted in common cell breakdown due to formation of dendritic structures on anode during cell cycling. The problems forced research and development of new intercalation materials for lithium batteries. In the eighties a new conception of lithium cell was proposed (so called Li-ion

© 2012 Molenda, licensee InTech. This is an open access chapter 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. © 2012 Molenda, licensee InTech. This is a paper 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.

batteries or "rocking-chair batteries"), which consisted of application of two different lithium intercalation compounds as anode and cathode materials [2-4]. As anode material a graphite intercalated with lithium was used while cathode materials were based on layered 3d transition metal oxides.

C/Li2MnSiO4 Nanocomposite Cathode Material for Li-Ion Batteries 63

An alternative material for cathode based on layered oxides LiMyCo1-yO2 (M=Ni, Mn, Fe) is LiMn2O4 spinel. LiMn2O4 reveals a little lower reversible capacity 120 mAh/g at working potential 4 V, but the material is distinctly cheaper and nontoxic. However, application of spinel as cathode material in commercial Li-ion batteries is retarded by phase transition observed near room temperature, i.e. battery operation temperature, and related to Jahn-Teller distortion of Mn3+ ions, what resulted in capacity fading. Stabilization of cubic spinel structure is possible by controlled formation of cationic defects [6], by lithium [7] or 3d metal substitution [8] into spinel lattice as well as by isoelectronic sulfur substitution [9, 10]. Instability of high oxidation state of transition metal in spinel structure observed in charged state of cathode leads to oxygen evolution, similarly to the layered oxide cathodes, and reaction with electrolyte. Application of spinel based materials is limited to cheap battery

An interesting and very promising group of insertion materials are LiMXO4 (M= metal 3d, X=S, P, As, Mo, W) type compounds [12], with LiFePO4 among them. LiFePO4 lithium iron phosphate of olivine structure is chemically and thermally stable material with relatively high gravimetric capacity 170 mAh/g at working potential 3.5 V. The material is cheap, nontoxic and environmental friendly. Its high chemical stability towards electrolyte, related to strong P-O bonds, significantly improves safety of LIBs. However, very low electrical conductivity of LiFePO4 system (10-9 S/cm @RT) requires application of carbon coatings and

**3. Orthosilicates Li2MSiO4 – New high capacity cathode materials**

Application of orthosilicates Li2MSiO4 (M=Fe, Mn, Co, Ni) compounds as insertion materials for LIBs was firstly proposed by Prof. Goodenough [12, 15]. The materials mainly crystallizes in orthorhombic system of Pmn21 space group in olivine structure [16-18]. The Li2MSiO4 olivine structure may be described as wavy layers of [SiMO4]∞ on *ac* axis plane and connected along *b* axis by LiO4 tetrahedra [16]. Within the layers every tetrahedron SiO4 shares its corner with four next MO4 tetrahedra. Lithium ions occupy the tetrahedral sites (LiO4) between two layers and share 3 and 1 oxygen atoms with the layers. In fact, diffusion of lithium ions in this structure is possible only through the canals formed by LiO4 tetrahedra. Li2MSiO4 silicates reveal a possibility of reversible insertion of two lithium ions per molecule, so this leads to exchange of two electrons by transition metal. As a results the silicates reveal very high theoretical gravimetric capacity, up to 330 mA/g. However, structural limitation due to sharing of LiO4 between two wavy layers, should affect a little the capacity. Calculations of electrochemical potential of lithium insertion/deinsertion show

Li2M2+SiO4 → □LiM3+SiO4 + Li+ (3)

**2.2. Spinel LiMn2O4 and related systems**

**2.3. Olivine LiFePO4 cathode material**

composite formation [13, 14].

a two stage process:

packs for EV [11].

## **2.1. Layered LiCoO2 oxide and related systems**

The first LixC6/Li+/Li1-xCoO2 Li-ion battery system was commercialized in 1993 by Sony Co. In Fig. 1 a working mechanism during discharge cycle of Li-ion cell is presented.

**Figure 1.** Working mechanism of Li-ion batteries (discharge cycle).

The electrochemical reaction at the graphite anode side can be written as (1):

$$\mathbf{C}s + \mathbf{x}\mathbf{L}\mathbf{i}^{\star} + \mathbf{x}\mathbf{e}^{\star} \leftrightarrow \mathbf{L}\mathbf{i}\mathbf{e}\mathbf{C}s \tag{1}$$

and suitably at LiCoO2 cathode side (2):

$$\text{LiCoO} \leftrightarrow \text{Li} \lor \text{CoO} \vdash \text{xLi}^{+} + \text{xe}^{-} \tag{2}$$

Commercially available LIBs based on layered lithium cobalt oxide (LiCoO2) or related systems (LiNiyCo1-yO2, LiMnyCo1-yO2, LiMn1/3Ni1/3Co1/3O2) reveal reversible capacity 130-150 mAh/g and working potential 3.6-3.7 V. The oxide materials are expensive and toxic due to cobalt content and are unstable in an overcharged state, thus the safety is strongly affected. This is related to strong oxidizing behavior of the charged layered oxide cathode in contact with organic electrolyte what may lead to combustion or even explosion [4, 5]. Unfortunately, this effect may be also increased by application of nanosized materials with high surface area.

#### **2.2. Spinel LiMn2O4 and related systems**

62 Composites and Their Properties

3d transition metal oxides.

**2.1. Layered LiCoO2 oxide and related systems**

**Figure 1.** Working mechanism of Li-ion batteries (discharge cycle).

LiCoO2 ↔Li1-xCoO2 + xLi+ + xe-

and suitably at LiCoO2 cathode side (2):

high surface area.

The electrochemical reaction at the graphite anode side can be written as (1):

C6 + xLi+ + xe- ↔ LixC6 (1)

Commercially available LIBs based on layered lithium cobalt oxide (LiCoO2) or related systems (LiNiyCo1-yO2, LiMnyCo1-yO2, LiMn1/3Ni1/3Co1/3O2) reveal reversible capacity 130-150 mAh/g and working potential 3.6-3.7 V. The oxide materials are expensive and toxic due to cobalt content and are unstable in an overcharged state, thus the safety is strongly affected. This is related to strong oxidizing behavior of the charged layered oxide cathode in contact with organic electrolyte what may lead to combustion or even explosion [4, 5]. Unfortunately, this effect may be also increased by application of nanosized materials with

(2)

batteries or "rocking-chair batteries"), which consisted of application of two different lithium intercalation compounds as anode and cathode materials [2-4]. As anode material a graphite intercalated with lithium was used while cathode materials were based on layered

The first LixC6/Li+/Li1-xCoO2 Li-ion battery system was commercialized in 1993 by Sony Co.

In Fig. 1 a working mechanism during discharge cycle of Li-ion cell is presented.

An alternative material for cathode based on layered oxides LiMyCo1-yO2 (M=Ni, Mn, Fe) is LiMn2O4 spinel. LiMn2O4 reveals a little lower reversible capacity 120 mAh/g at working potential 4 V, but the material is distinctly cheaper and nontoxic. However, application of spinel as cathode material in commercial Li-ion batteries is retarded by phase transition observed near room temperature, i.e. battery operation temperature, and related to Jahn-Teller distortion of Mn3+ ions, what resulted in capacity fading. Stabilization of cubic spinel structure is possible by controlled formation of cationic defects [6], by lithium [7] or 3d metal substitution [8] into spinel lattice as well as by isoelectronic sulfur substitution [9, 10]. Instability of high oxidation state of transition metal in spinel structure observed in charged state of cathode leads to oxygen evolution, similarly to the layered oxide cathodes, and reaction with electrolyte. Application of spinel based materials is limited to cheap battery packs for EV [11].

## **2.3. Olivine LiFePO4 cathode material**

An interesting and very promising group of insertion materials are LiMXO4 (M= metal 3d, X=S, P, As, Mo, W) type compounds [12], with LiFePO4 among them. LiFePO4 lithium iron phosphate of olivine structure is chemically and thermally stable material with relatively high gravimetric capacity 170 mAh/g at working potential 3.5 V. The material is cheap, nontoxic and environmental friendly. Its high chemical stability towards electrolyte, related to strong P-O bonds, significantly improves safety of LIBs. However, very low electrical conductivity of LiFePO4 system (10-9 S/cm @RT) requires application of carbon coatings and composite formation [13, 14].

## **3. Orthosilicates Li2MSiO4 – New high capacity cathode materials**

Application of orthosilicates Li2MSiO4 (M=Fe, Mn, Co, Ni) compounds as insertion materials for LIBs was firstly proposed by Prof. Goodenough [12, 15]. The materials mainly crystallizes in orthorhombic system of Pmn21 space group in olivine structure [16-18]. The Li2MSiO4 olivine structure may be described as wavy layers of [SiMO4]∞ on *ac* axis plane and connected along *b* axis by LiO4 tetrahedra [16]. Within the layers every tetrahedron SiO4 shares its corner with four next MO4 tetrahedra. Lithium ions occupy the tetrahedral sites (LiO4) between two layers and share 3 and 1 oxygen atoms with the layers. In fact, diffusion of lithium ions in this structure is possible only through the canals formed by LiO4 tetrahedra. Li2MSiO4 silicates reveal a possibility of reversible insertion of two lithium ions per molecule, so this leads to exchange of two electrons by transition metal. As a results the silicates reveal very high theoretical gravimetric capacity, up to 330 mA/g. However, structural limitation due to sharing of LiO4 between two wavy layers, should affect a little the capacity. Calculations of electrochemical potential of lithium insertion/deinsertion show a two stage process:

$$\text{Li} \text{\textbullet} \text{M}^{2+} \text{\textbullet} \text{\textbullet} \to \text{Li} \text{\textbullet} \text{\textbullet} \text{\textbullet} \text{\textbullet} \text{\textbullet} \text{\textbullet} \tag{3}$$

$$\rm YLiM^{3+} \rm SiO\_4 \rightarrow \rm Y\_2M^{4+} \rm SiO\_4 + Li^\* \tag{4}$$

C/Li2MnSiO4 Nanocomposite Cathode Material for Li-Ion Batteries 65

metal acetates. Prepared mixture was heated to 60 C and few drops of concentrated hydrochloric acid was added to initiate polymerization of metal citrates using ethylene glycol and TEOS. Reaction was conducted for 24h in close reactor. Obtained gel was aged for 3 days at 60 C in close reactor (Ar atmosphere) and for 3 days at 60 C in an air-drier

Thermogravimetric analysis (TGA) coupled with simultaneous differential thermal analysis (SDTA) and mass spectrometry evolved gas analysis (MS-EGA) of precursor were performed in Mettler-Toledo 851e thermo-analyzer using 150 μl corundum crucibles under flow of air/argon (80 ml/min), within temperature range 20–800 °C with heating rate of 10 °C/min. The simultaneous MS-EGA was performed in on-line joined quadruple mass spectrometer (QMS) (Thermostar-Balzers). The 17, 18 and 44 m/z mass lines, ascribed to OH, H2O and CO2 species respectively, were collected during the TGA experiments. TGA of the

**Figure 2.** Thermal analysis (TGA/DTG/SDTA) conducted under air atmosphere (a) with evolved gas

**Figure 3.** Thermal analysis (TGA/DTG/SDTA) of Li2MnSiO4 precursor performed under inert

Li2MnSiO4 precursor are shown in the Fig. 2 and Fig. 3.

analysis (EGA-QMS) (b) of Li2MnSiO4 precursor.

atmosphere (Ar 5.0).

(Air atmosphere).

Depending on transition metal potential of the reaction (3) vary from 3.2 V (for Fe) to 4.1-4.4 V (for Mn, Co, Ni), while the potential of reaction (4) is in range 4.5-5.0 V. Deinsertion of the second lithium ion requires applying high potential above 4.5 V, and this is a challenge for electrolyte. Orthosilicates Li2MSiO4 (M=Fe, Mn, Co, Ni) materials reveal very low electrical conductivity (10-12 - 10-15 S/cm @RT) and carbon coating of the materials is required. On the other hand, downsizing of material grains should improve electrochemical performance [18]. Very strong covalent bonding of Si-O results in high chemical stability towards electrolyte, strongly increasing the safety of LIBs based on silicates. The materials are nontoxic, environmental friendly and cheap. The properties mentioned above provide a challenge for developing a composite cathode materials based on Li2MSiO4 orthosilicates.

## **4. Li2MnSiO4 nanostructured cathode material**

Li2MnSiO4 is a member of dilithiumorthosilicates Li2MSiO4 (M = Fe, Mn, Co) family, thanks to strong covalent Si-O bond, it shows high thermal and chemical stability. High theoretical capacity 333 mAh/g, low production costs, safety and optimal working potential make them an attractive cathode material. Very low electrical conductivity (10-12 - 10-15 S/cm @RT) can be improved by coating with conductive carbon layers (CCL) and by grains size downsizing [18-20]. The properties of Li2MnSiO4 material comes to a conclusion that cathode material for LIBs based on this compound should be prepared as C/Li2MnSiO4 nanocomposite. Thus, the special preparation techniques have to be applied in terms to obtain nanocrystaline grains of Li2MnSiO4 cathode material coated by conductive carbon layers (CCL). During last few years several different technics of Li2MnSiO4 synthesis were proposed. Hydrothermal synthesis [21-24] as well as solid-state reactions [25-28] can lead to one phase product but the control of the grain size is significantly limited. Sol-gel method is one of the soft chemistry techniques which can be used in synthesis of nanosized lithium orthosilicates [29-33]. Sol-gel processes, especially Pechini's method is a simple technic, characterized by low cost and low temperature of treatment, resulting in homogenous, high purity materials.

### **4.1. Preparation of nanostructured Li2MnSiO4**

Li2MnSiO4 was produced using sol-gel synthesis – Pechini type. Starting reagents were: lithium acetate dihydrate (Aldrich), manganese acetate tetrahydrate (Aldrich), tetraethoxysilane (TEOS) (98%, Aldrich) as a source of silicon, ethylene glycol (POCh), citric acid (POCh) and ethanol (POCh). Thanks to chelating metal ions in solution by citric acid the cations can be mixed at the molecular level and the stoichiometric composition can be achived. The reactants were mixed in a molar ratio 1:1:18:6:4:16 - Mn:Si:C2H6O2:C6H8O7:C2H5OH:H2O. Based on previous studies it was affirmed, that using 20% excess of lithium acetate leads to one-phase product. All reagents were dissolved in glass reactor under constant argon flow (Ar 5.7). As a solvent, only stoichiometric amount of distilled water was used. Heating water to 35 C assure fast and complete dissolution of metal acetates. Prepared mixture was heated to 60 C and few drops of concentrated hydrochloric acid was added to initiate polymerization of metal citrates using ethylene glycol and TEOS. Reaction was conducted for 24h in close reactor. Obtained gel was aged for 3 days at 60 C in close reactor (Ar atmosphere) and for 3 days at 60 C in an air-drier (Air atmosphere).

64 Composites and Their Properties

LiM3+SiO4 → 2M4+SiO4 + Li+ (4)

Depending on transition metal potential of the reaction (3) vary from 3.2 V (for Fe) to 4.1-4.4 V (for Mn, Co, Ni), while the potential of reaction (4) is in range 4.5-5.0 V. Deinsertion of the second lithium ion requires applying high potential above 4.5 V, and this is a challenge for electrolyte. Orthosilicates Li2MSiO4 (M=Fe, Mn, Co, Ni) materials reveal very low electrical conductivity (10-12 - 10-15 S/cm @RT) and carbon coating of the materials is required. On the other hand, downsizing of material grains should improve electrochemical performance [18]. Very strong covalent bonding of Si-O results in high chemical stability towards electrolyte, strongly increasing the safety of LIBs based on silicates. The materials are nontoxic, environmental friendly and cheap. The properties mentioned above provide a challenge for developing a composite cathode materials based on Li2MSiO4 orthosilicates.

Li2MnSiO4 is a member of dilithiumorthosilicates Li2MSiO4 (M = Fe, Mn, Co) family, thanks to strong covalent Si-O bond, it shows high thermal and chemical stability. High theoretical capacity 333 mAh/g, low production costs, safety and optimal working potential make them an attractive cathode material. Very low electrical conductivity (10-12 - 10-15 S/cm @RT) can be improved by coating with conductive carbon layers (CCL) and by grains size downsizing [18-20]. The properties of Li2MnSiO4 material comes to a conclusion that cathode material for LIBs based on this compound should be prepared as C/Li2MnSiO4 nanocomposite. Thus, the special preparation techniques have to be applied in terms to obtain nanocrystaline grains of Li2MnSiO4 cathode material coated by conductive carbon layers (CCL). During last few years several different technics of Li2MnSiO4 synthesis were proposed. Hydrothermal synthesis [21-24] as well as solid-state reactions [25-28] can lead to one phase product but the control of the grain size is significantly limited. Sol-gel method is one of the soft chemistry techniques which can be used in synthesis of nanosized lithium orthosilicates [29-33]. Sol-gel processes, especially Pechini's method is a simple technic, characterized by low cost and low temperature of

Li2MnSiO4 was produced using sol-gel synthesis – Pechini type. Starting reagents were: lithium acetate dihydrate (Aldrich), manganese acetate tetrahydrate (Aldrich), tetraethoxysilane (TEOS) (98%, Aldrich) as a source of silicon, ethylene glycol (POCh), citric acid (POCh) and ethanol (POCh). Thanks to chelating metal ions in solution by citric acid the cations can be mixed at the molecular level and the stoichiometric composition can be achived. The reactants were mixed in a molar ratio 1:1:18:6:4:16 - Mn:Si:C2H6O2:C6H8O7:C2H5OH:H2O. Based on previous studies it was affirmed, that using 20% excess of lithium acetate leads to one-phase product. All reagents were dissolved in glass reactor under constant argon flow (Ar 5.7). As a solvent, only stoichiometric amount of distilled water was used. Heating water to 35 C assure fast and complete dissolution of

**4. Li2MnSiO4 nanostructured cathode material**

treatment, resulting in homogenous, high purity materials.

**4.1. Preparation of nanostructured Li2MnSiO4**

Thermogravimetric analysis (TGA) coupled with simultaneous differential thermal analysis (SDTA) and mass spectrometry evolved gas analysis (MS-EGA) of precursor were performed in Mettler-Toledo 851e thermo-analyzer using 150 μl corundum crucibles under flow of air/argon (80 ml/min), within temperature range 20–800 °C with heating rate of 10 °C/min. The simultaneous MS-EGA was performed in on-line joined quadruple mass spectrometer (QMS) (Thermostar-Balzers). The 17, 18 and 44 m/z mass lines, ascribed to OH, H2O and CO2 species respectively, were collected during the TGA experiments. TGA of the Li2MnSiO4 precursor are shown in the Fig. 2 and Fig. 3.

**Figure 2.** Thermal analysis (TGA/DTG/SDTA) conducted under air atmosphere (a) with evolved gas analysis (EGA-QMS) (b) of Li2MnSiO4 precursor.

**Figure 3.** Thermal analysis (TGA/DTG/SDTA) of Li2MnSiO4 precursor performed under inert atmosphere (Ar 5.0).

According to TGA curve (Fig. 2a) a complete decomposition of gel organic matrix occurs above 500 °C. Evolved gas analysis confirmed the disintegration of organic components. Amount of active material can be estimated at about 12 wt.% of the precursor. Basing on TGA results calcination conditions were chosen. Li2MnSiO4 precursor was calcined under Ar flow at 600, 700, 800 and 900 °C.

C/Li2MnSiO4 Nanocomposite Cathode Material for Li-Ion Batteries 67

Measurements of the specific surface area of the samples were performed in Micrometrics ASAP 2010 using BET isotherm method. About 500 mg of each sample was preliminary degassed at 250–300 °C for 3 h under pressure 0.26–0.4 Pa. Then, N2 sorption was performed at pressure of 8· 104 Pa. One of pore size distribution plot with the adsorption-desorption curves inset is presented in Fig. 5a. Exact values of specific surface area and average pores

**Figure 5.** Adsorption-desorption isotherm and pore diameter distribution of C/Li2MnSiO4 calcined at

Low temperature N2-adsorption (BET) measurements of C/Li2MnSiO4 composites show that specific surface area of samples decrease with increasing calcination temperature. Decrease of specific surface area is connected with graphitization of carbon during calcination at

> Carbon content

C/Li2MnSiO4@600 600 °C 34% 169 m2/g 50 Å 17 nm C/Li2MnSiO4@700 700 °C 32% 152 m2/g 72 Å 18 nm C/Li2MnSiO4@800 800 °C 32% 144 m2/g 73 Å 19 nm C/Li2MnSiO4@900 900 °C 35% 57 m2/g 73 Å 19 nm

**Table 1.** Morphology parameters of C/Li2MnSiO4 composites calcined at different temperatures.

Electrical conductivity was measured using the AC (33Hz) 4-probe method within temperature range of -40÷55 °C. The carbon coated composite powders were so elastic that the standard preparation of pellets was impossible. The fine powder samples were placed into a glass tube and pressed by a screw-press between parallel gold disc electrodes (∅=5 mm) till the measured resistance remained constant. The results of electrical conductivity measurements are gather in Fig. 6. All composites exhibit good electrical conductivity (up to 0,36 S/cm for C/Li2MnSiO4@900) in comparison with Li2MnSiO4 itself ( ̴10-15 S/cm at room

Specific surface area (BET)

Average pore dimension (BJH)

Crystallite size (from XRD)

600°C, b) TGA/DTG/SDTA curves of C/Li2MnSiO4 calcined at 600 °C.

Precursor calcination temperature

diameters are presented in table 1.

higher temperatures.

Sample name

#### **4.2. Properties of nanostructured Li2MnSiO4**

X-ray powder diffraction patterns of the samples were collected on BRUKER D2 PHASER using Cu Kα radiation = 1.5418 Å. In Fig. 4 XRD patterns of Li2MnSiO4 obtained at different temperatures (600, 700, 800 and 900 °C) are collected.

**Figure 4.** XRD patterns of Li2MnSiO4 after calcination at 600 °C, 700 °C, 800 °C and 900 °C. Li2MnSiO4 (*Pmn21* phase) diffraction lines positions are marked in the top of the figure.

In the sample calcined at 600 °C all diffraction lines can be attributed to pure Li2MnSiO4 phase (*Pmn21*). In other specimens obtained at higher temperatures, MnO and Li2SiO3 impurities starts to appear. Formation of MnO and Li2SiO3 in samples calcined at temperatures above 700 °C can be connected, with Li2MnSiO4 decomposition. Average crystallites sizes calculated from diffraction lines broadening based on Scherrer equation are in the range of 17-19 nm depending on calcination temperature of the sample (Tab. 1). The calcination under carefully selected conditions leads to a transformation of a part of organic compounds from the gel matrix into thick carbon layer covering the active material. The temperature programmed oxidation (TPO) performed using TGA equipment confirmed presence of carbon in samples. Fig. 5b presents exemplary TG/DTG/SDTA curves from TPO conducted under air flow in temperature range of 20-1000 °C. The amounts of carbon in samples calcined in different temperatures were calculated from TPO measurements and are collected in Table 1.

Measurements of the specific surface area of the samples were performed in Micrometrics ASAP 2010 using BET isotherm method. About 500 mg of each sample was preliminary degassed at 250–300 °C for 3 h under pressure 0.26–0.4 Pa. Then, N2 sorption was performed at pressure of 8· 104 Pa. One of pore size distribution plot with the adsorption-desorption curves inset is presented in Fig. 5a. Exact values of specific surface area and average pores diameters are presented in table 1.

66 Composites and Their Properties

collected in Table 1.

flow at 600, 700, 800 and 900 °C.

**4.2. Properties of nanostructured Li2MnSiO4**

temperatures (600, 700, 800 and 900 °C) are collected.

According to TGA curve (Fig. 2a) a complete decomposition of gel organic matrix occurs above 500 °C. Evolved gas analysis confirmed the disintegration of organic components. Amount of active material can be estimated at about 12 wt.% of the precursor. Basing on TGA results calcination conditions were chosen. Li2MnSiO4 precursor was calcined under Ar

X-ray powder diffraction patterns of the samples were collected on BRUKER D2 PHASER using Cu Kα radiation = 1.5418 Å. In Fig. 4 XRD patterns of Li2MnSiO4 obtained at different

**Figure 4.** XRD patterns of Li2MnSiO4 after calcination at 600 °C, 700 °C, 800 °C and 900 °C. Li2MnSiO4

In the sample calcined at 600 °C all diffraction lines can be attributed to pure Li2MnSiO4 phase (*Pmn21*). In other specimens obtained at higher temperatures, MnO and Li2SiO3 impurities starts to appear. Formation of MnO and Li2SiO3 in samples calcined at temperatures above 700 °C can be connected, with Li2MnSiO4 decomposition. Average crystallites sizes calculated from diffraction lines broadening based on Scherrer equation are in the range of 17-19 nm depending on calcination temperature of the sample (Tab. 1). The calcination under carefully selected conditions leads to a transformation of a part of organic compounds from the gel matrix into thick carbon layer covering the active material. The temperature programmed oxidation (TPO) performed using TGA equipment confirmed presence of carbon in samples. Fig. 5b presents exemplary TG/DTG/SDTA curves from TPO conducted under air flow in temperature range of 20-1000 °C. The amounts of carbon in samples calcined in different temperatures were calculated from TPO measurements and are

(*Pmn21* phase) diffraction lines positions are marked in the top of the figure.

**Figure 5.** Adsorption-desorption isotherm and pore diameter distribution of C/Li2MnSiO4 calcined at 600°C, b) TGA/DTG/SDTA curves of C/Li2MnSiO4 calcined at 600 °C.

Low temperature N2-adsorption (BET) measurements of C/Li2MnSiO4 composites show that specific surface area of samples decrease with increasing calcination temperature. Decrease of specific surface area is connected with graphitization of carbon during calcination at higher temperatures.

**Table 1.** Morphology parameters of C/Li2MnSiO4 composites calcined at different temperatures.

Electrical conductivity was measured using the AC (33Hz) 4-probe method within temperature range of -40÷55 °C. The carbon coated composite powders were so elastic that the standard preparation of pellets was impossible. The fine powder samples were placed into a glass tube and pressed by a screw-press between parallel gold disc electrodes (∅=5 mm) till the measured resistance remained constant. The results of electrical conductivity measurements are gather in Fig. 6. All composites exhibit good electrical conductivity (up to 0,36 S/cm for C/Li2MnSiO4@900) in comparison with Li2MnSiO4 itself ( ̴10-15 S/cm at room temperature - RT). It can be observed that temperature dependence of conductivity for composites calcined at higher temperatures (700, 800 and 900 °C) seem unaffected, by temperature (metallic-like behavior). Conductivity value and its invariability against temperature indicate that carbon layers consist of graphite-like domains. In case of C/Li2MnSiO4@600°C the carbon layers is more disordered and probably consist of an activated-like carbon. Those results are consistent with BET measurements in case of graphitization process occurring at higher temperatures.

C/Li2MnSiO4 Nanocomposite Cathode Material for Li-Ion Batteries 69

**Figure 7.** TEM micrographs of C/Li2MnSiO4@600. Micrographs a), c) and d) present bright field images of C/Li2MnSiO4@600 composite in different magnification; b) microstructure image observed in STEM-HAADF with EDS analysis (b1; b2) from marked points; e) high resolution micrograph (HREM) of a

single grain and IFFT image f) from the same region.

**Figure 6.** Electrical conductivity measurement of C/Li2MnSiO4 (temp. range: -20 to 40°C). Dash line represent room temperature (RT).

Selected samples were investigated using transmission electron microscope (TEM) (Fig. 7 and Fig. 8). Micrographs were collected on TECNAI G2 F20 (200 kV) coupled with an energy dispersive X-ray spectrometer (EDAX).

TEM micrographs reveal well dispersed lithium manganese orthosilicate grains (dark grey and black dots) in the carbon matrix. Figures 7a; 7c; 7d present C/Li2MnSiO4@600 at different magnifications. Fig. 7b shows STEM-HAADF (*Scanning Transmission Electron Microscopy – High Angular Annular Dark Field*) image of the C/Li2MnSiO4@600 composite. Energy dispersive X-ray spectroscopy (EDS) (Fig. 7b1; 7b2) confirmed presence of lithium manganese silicate in the spots marked in the micrograph 7b (Cu peaks in all EDS spectra originate from TEM copper grid). High resolution electron microscopy image (HREM) shows single grain of Li2MnSiO4 (Fig. 7e). Corresponding to that IFFT (*Inverse Fast Furrier Transform*) image (Fig. 7f) clearly shows a crystalline structure of obtained material which can be identified as Li2MnSiO4. Grains in the composite have sizes in a range of 5-10 nm. Microstructure of a C/Li2MnSiO4 composite calcined at 700 °C is shown in Fig. 8. C/Li2MnSiO4@700 °C composite has a bimodal distribution of a crystallite size. TEM micrographs show presence of small (5-10 nm) Li2MnSiO4 crystallites, analogues of C/Li2MnSiO4@600 °C and a bigger, well crystallized silicates grains, in a range of 50-150 nm (Fig. 8a, c, d). Both types of crystallites in the sample have the same composition which is confirmed by EDS analysis presented in fig. 8b1, b2, b3.

represent room temperature (RT).

energy dispersive X-ray spectrometer (EDAX).

confirmed by EDS analysis presented in fig. 8b1, b2, b3.

graphitization process occurring at higher temperatures.

temperature - RT). It can be observed that temperature dependence of conductivity for composites calcined at higher temperatures (700, 800 and 900 °C) seem unaffected, by temperature (metallic-like behavior). Conductivity value and its invariability against temperature indicate that carbon layers consist of graphite-like domains. In case of C/Li2MnSiO4@600°C the carbon layers is more disordered and probably consist of an activated-like carbon. Those results are consistent with BET measurements in case of

**Figure 6.** Electrical conductivity measurement of C/Li2MnSiO4 (temp. range: -20 to 40°C). Dash line

Selected samples were investigated using transmission electron microscope (TEM) (Fig. 7 and Fig. 8). Micrographs were collected on TECNAI G2 F20 (200 kV) coupled with an

TEM micrographs reveal well dispersed lithium manganese orthosilicate grains (dark grey and black dots) in the carbon matrix. Figures 7a; 7c; 7d present C/Li2MnSiO4@600 at different magnifications. Fig. 7b shows STEM-HAADF (*Scanning Transmission Electron Microscopy – High Angular Annular Dark Field*) image of the C/Li2MnSiO4@600 composite. Energy dispersive X-ray spectroscopy (EDS) (Fig. 7b1; 7b2) confirmed presence of lithium manganese silicate in the spots marked in the micrograph 7b (Cu peaks in all EDS spectra originate from TEM copper grid). High resolution electron microscopy image (HREM) shows single grain of Li2MnSiO4 (Fig. 7e). Corresponding to that IFFT (*Inverse Fast Furrier Transform*) image (Fig. 7f) clearly shows a crystalline structure of obtained material which can be identified as Li2MnSiO4. Grains in the composite have sizes in a range of 5-10 nm. Microstructure of a C/Li2MnSiO4 composite calcined at 700 °C is shown in Fig. 8. C/Li2MnSiO4@700 °C composite has a bimodal distribution of a crystallite size. TEM micrographs show presence of small (5-10 nm) Li2MnSiO4 crystallites, analogues of C/Li2MnSiO4@600 °C and a bigger, well crystallized silicates grains, in a range of 50-150 nm (Fig. 8a, c, d). Both types of crystallites in the sample have the same composition which is

**Figure 7.** TEM micrographs of C/Li2MnSiO4@600. Micrographs a), c) and d) present bright field images of C/Li2MnSiO4@600 composite in different magnification; b) microstructure image observed in STEM-HAADF with EDS analysis (b1; b2) from marked points; e) high resolution micrograph (HREM) of a single grain and IFFT image f) from the same region.

C/Li2MnSiO4 Nanocomposite Cathode Material for Li-Ion Batteries 71

STEM-HAADF image (Fig. 8b) from the same region as a bright field Fig. 8a shows points from where EDS analysis was conducted. All of lithium manganese orthosilicate crystallites in C/Li2MnSiO4@700 °C are well dispersed and fully covered in amorphous carbon. HREM image in Fig. 8d1 with IFFT in Fig. 8d2 shows crystalline structure of

Nevertheless obtained composites show homogeneous distribution of particles in carbon matrix and exhibit good electrical conductivity they work poorly in a battery cell (see paragraph 5.2). Thickness of primary carbon coating on active material grains strongly

Preparation of composites with well define morphology of carbon layers and optimal carbon content was achieved by burning out the primary carbon and recoating Li2MnSiO4 nanosized grains with conductive carbon layers (CCL/Li2MnSiO4 composites). Burning out of primary carbon formed in the sample during synthesis process was carried through calcination of C/Li2MnSiO4@600 and C/Li2MnSiO4@700 under air flow at 300 °C for 3 h. CCL/Li2MnSiO4 composites were produced by wet polymer precursor deposition on active material grains and subsequent controlled pyrolysis [14, 20, 34]. Poly-N-vinylformamide (PNVF) obtained by radical-free polymerization of Nvinylformamide (Aldrich) with pyromellitic acid (PMA) additive (5-10 wt%) was used as a carbon polymer precursor [34]. To achieve an impregnation, Li2MnSiO4 grains were suspended in water polymer solution (8-15 wt%). Finally, samples were dried up in an air drier at 90 °C for 24 h. Prepared samples were pyrolyzed at 600 °C for 6 h under inert

Burning out primary carbon from the surface of lithium manganese silicate in air atmosphere leads to partial decomposition of active material. Fig. 9 shows XRD patterns of C/Li2MnSiO4@600, Li2MnSiO4@600 without carbon and CCL/Li2MnSiO4@600 composite after

In diffraction patterns of Li2MnSiO4@600 it is clearly visible that after burning out primary carbon layer new phase appears in the sample (LiMn2O4). Proposed carbon coating process can reverse decomposition of Li2MnSiO4. After coating Li2MnSiO4 (*Pmn21*) is a dominant phase in CCL/Li2MnSiO4@600 and all the lines, except for very low intensity line around 19°, can be associated with Li2MnSiO4 phase. CCL preparation procedure influence active material grain size. Average crystallite size calculated using Scherer equation in CCL/Li2MnSiO4@600 is ca. 25 nm. TEM analysis confirms increase of crystallites diameters. Li2MnSiO4 grains observed under TEM are in the range 25-75 nm. TEM micrographs of

Li2MnSiO4 grain.

atmosphere (Ar).

coating with CCL from polymer precursor.

CCL/Li2MnSiO4@600 are presented in Fig. 10.

**5. C/Li2MnSiO4 composite cathode material**

limits the electrochemical performance of composite.

**5.1. CCL/Li2MnSiO4 composite preparation and properties**

**Figure 8.** TEM micrographs of C/Li2MnSiO4@700. Micrographs a), c) and d) presents bright field images of C/Li2MnSiO4@700 composite in different magnification; b) microstructure image observed in STEM-HAADF with EDS analysis (b1; b2; b3) from selected points; d1) high resolution micrograph (HREM) of region marked in Fig. 8d; d2) IFFT image from square marked in d1).

STEM-HAADF image (Fig. 8b) from the same region as a bright field Fig. 8a shows points from where EDS analysis was conducted. All of lithium manganese orthosilicate crystallites in C/Li2MnSiO4@700 °C are well dispersed and fully covered in amorphous carbon. HREM image in Fig. 8d1 with IFFT in Fig. 8d2 shows crystalline structure of Li2MnSiO4 grain.

## **5. C/Li2MnSiO4 composite cathode material**

70 Composites and Their Properties

**Figure 8.** TEM micrographs of C/Li2MnSiO4@700. Micrographs a), c) and d) presents bright field images of C/Li2MnSiO4@700 composite in different magnification; b) microstructure image observed in STEM-HAADF with EDS analysis (b1; b2; b3) from selected points; d1) high resolution micrograph (HREM) of

region marked in Fig. 8d; d2) IFFT image from square marked in d1).

Nevertheless obtained composites show homogeneous distribution of particles in carbon matrix and exhibit good electrical conductivity they work poorly in a battery cell (see paragraph 5.2). Thickness of primary carbon coating on active material grains strongly limits the electrochemical performance of composite.

## **5.1. CCL/Li2MnSiO4 composite preparation and properties**

Preparation of composites with well define morphology of carbon layers and optimal carbon content was achieved by burning out the primary carbon and recoating Li2MnSiO4 nanosized grains with conductive carbon layers (CCL/Li2MnSiO4 composites). Burning out of primary carbon formed in the sample during synthesis process was carried through calcination of C/Li2MnSiO4@600 and C/Li2MnSiO4@700 under air flow at 300 °C for 3 h. CCL/Li2MnSiO4 composites were produced by wet polymer precursor deposition on active material grains and subsequent controlled pyrolysis [14, 20, 34]. Poly-N-vinylformamide (PNVF) obtained by radical-free polymerization of Nvinylformamide (Aldrich) with pyromellitic acid (PMA) additive (5-10 wt%) was used as a carbon polymer precursor [34]. To achieve an impregnation, Li2MnSiO4 grains were suspended in water polymer solution (8-15 wt%). Finally, samples were dried up in an air drier at 90 °C for 24 h. Prepared samples were pyrolyzed at 600 °C for 6 h under inert atmosphere (Ar).

Burning out primary carbon from the surface of lithium manganese silicate in air atmosphere leads to partial decomposition of active material. Fig. 9 shows XRD patterns of C/Li2MnSiO4@600, Li2MnSiO4@600 without carbon and CCL/Li2MnSiO4@600 composite after coating with CCL from polymer precursor.

In diffraction patterns of Li2MnSiO4@600 it is clearly visible that after burning out primary carbon layer new phase appears in the sample (LiMn2O4). Proposed carbon coating process can reverse decomposition of Li2MnSiO4. After coating Li2MnSiO4 (*Pmn21*) is a dominant phase in CCL/Li2MnSiO4@600 and all the lines, except for very low intensity line around 19°, can be associated with Li2MnSiO4 phase. CCL preparation procedure influence active material grain size. Average crystallite size calculated using Scherer equation in CCL/Li2MnSiO4@600 is ca. 25 nm. TEM analysis confirms increase of crystallites diameters. Li2MnSiO4 grains observed under TEM are in the range 25-75 nm. TEM micrographs of CCL/Li2MnSiO4@600 are presented in Fig. 10.

C/Li2MnSiO4 Nanocomposite Cathode Material for Li-Ion Batteries 73

**Figure 10.** TEM micrographs of CCL/Li2MnSiO4@600. Micrographs a), c) and d) presents bright field images of CCL/Li2MnSiO4@600 composite; b) microstructure image observed in STEM-HAADF with EDS analysis (b1) from selected point; e) HREM micrograph of carbon coating on the active material

grain surface.

**Figure 9.** XRD patterns of C/Li2MnSiO4@600, Li2MnSiO4@600 and CCL/Li2MnSiO4@600.

Bright field micrographs (Fig. 10a, c, d) shows crystallites of Li2MnSiO4 covered with conductive carbon layers (CCL). Formed carbon coatings adhere well to the surface of active material grains, no voids are visible at the CCL/Li2MnSiO4 interface (Fig. 10e). Silicate grains are uniformly covered with approximately 4-5 nm thick CCL. EDS analysis once again confirm presence of lithium manganese silicate in the composite (Fig. 10b, b1). Carbon content in samples was calculated from TPO measurements and they are displayed in Table 2.

**Table 2.** Carbon content of CCL/Li2MnSiO4 composites.

Table 2.

**Figure 9.** XRD patterns of C/Li2MnSiO4@600, Li2MnSiO4@600 and CCL/Li2MnSiO4@600.

Sample name Precursor calcination

**Table 2.** Carbon content of CCL/Li2MnSiO4 composites.

Bright field micrographs (Fig. 10a, c, d) shows crystallites of Li2MnSiO4 covered with conductive carbon layers (CCL). Formed carbon coatings adhere well to the surface of active material grains, no voids are visible at the CCL/Li2MnSiO4 interface (Fig. 10e). Silicate grains are uniformly covered with approximately 4-5 nm thick CCL. EDS analysis once again confirm presence of lithium manganese silicate in the composite (Fig. 10b, b1). Carbon content in samples was calculated from TPO measurements and they are displayed in

temperature

<EMAIL_ADDRESS>600 °C 7.5% CCL/Li2MnSiO4@600\_12% 600 °C 12% CCL/Li2MnSiO4@700\_5% 700 °C 5% CCL/Li2MnSiO4@700\_10% 700 °C 10%

Carbon content

**Figure 10.** TEM micrographs of CCL/Li2MnSiO4@600. Micrographs a), c) and d) presents bright field images of CCL/Li2MnSiO4@600 composite; b) microstructure image observed in STEM-HAADF with EDS analysis (b1) from selected point; e) HREM micrograph of carbon coating on the active material grain surface.

## **5.2. Electrochemical properties of C/Li2MnSiO4 composite cathode**

The charge-discharge cycling studies of Li/Li+/(C/Li2MnSiO4) cells were conducted in a four electrode configuration using CR2032 assembly between 2.7 and 4.7 V at C/200 rate at room temperature. LiPF6 solution 1M in EC/DEC (1:1) was used in the cells as an electrolyte. The galvanostatic measurements were carried out using ATLAS 0961 MBI test system. Chargedischarge tests were conducted for all CCL composites listed in table 2, composites with primary carbon (C/Li2MnSiO4@600, C/Li2MnSiO4@700 and C/Li2MnSiO4@800) and standard composites (Li2MnSiO4@600\_CB and Li2MnSiO4@700\_CB). Standard composites were prepared by mixing Li2MnSiO4 powder with commercial carbon additive – carbon black (CB). 15 wt.% of carbon was used. Results collected from charge/discharge tests are presented in fig. 11-16.

C/Li2MnSiO4 Nanocomposite Cathode Material for Li-Ion Batteries 75

**Figure 12.** Cell cycling behavior of standard composites Li2MnSiO4 + carbon black (15% carbon

**Figure 13.** Cell cycling behavior of CCL/Li2MnSiO4@600 (7.5% carbon content)

content)

C/Li2MnSiO4@600 sample did not show any reversible capacity (Fig. 11a). Lack of electrochemical activity of C/Li2MnSiO4@600 is connected with too high carbon loading. High carbon content (34%) is responsible for limiting of ionic conductivity in the composite and for surface polarization. Galvanostatic cycling studies of C/Li2MnSiO4@700 and C/Li2MnSiO4@800 revealed reversible capacity in a range of 30 mAh·g-1. Fig. 11b show charge and discharge capacity for 7 cycles of C/Li2MnSiO4@700 composite. After first four cycles reversible capacity stabilizes at about 30 mAh·g-1. Very poor performance of this sample is also connected with high amount of carbon in prepared composite (32%).

Standard composites obtained by mixing active silicate materials with carbon black (15%) show extremely low reversible capacity as well (Fig. 12a and 12b). In this case, carbon additive does not provide sufficient electrical contact between active material grains. Due to this fact, even under low C/200 rate samples performed very poorly in charge/discharge tests.

**Figure 11.** Cell cycling behavior of: a) C/Li2MnSiO4@600, b) C/Li2MnSiO4@700.

presented in fig. 11-16.

prepared composite (32%).

tests.

**5.2. Electrochemical properties of C/Li2MnSiO4 composite cathode**

The charge-discharge cycling studies of Li/Li+/(C/Li2MnSiO4) cells were conducted in a four electrode configuration using CR2032 assembly between 2.7 and 4.7 V at C/200 rate at room temperature. LiPF6 solution 1M in EC/DEC (1:1) was used in the cells as an electrolyte. The galvanostatic measurements were carried out using ATLAS 0961 MBI test system. Chargedischarge tests were conducted for all CCL composites listed in table 2, composites with primary carbon (C/Li2MnSiO4@600, C/Li2MnSiO4@700 and C/Li2MnSiO4@800) and standard composites (Li2MnSiO4@600\_CB and Li2MnSiO4@700\_CB). Standard composites were prepared by mixing Li2MnSiO4 powder with commercial carbon additive – carbon black (CB). 15 wt.% of carbon was used. Results collected from charge/discharge tests are

C/Li2MnSiO4@600 sample did not show any reversible capacity (Fig. 11a). Lack of electrochemical activity of C/Li2MnSiO4@600 is connected with too high carbon loading. High carbon content (34%) is responsible for limiting of ionic conductivity in the composite and for surface polarization. Galvanostatic cycling studies of C/Li2MnSiO4@700 and C/Li2MnSiO4@800 revealed reversible capacity in a range of 30 mAh·g-1. Fig. 11b show charge and discharge capacity for 7 cycles of C/Li2MnSiO4@700 composite. After first four cycles reversible capacity stabilizes at about 30 mAh·g-1. Very poor performance of this sample is also connected with high amount of carbon in

Standard composites obtained by mixing active silicate materials with carbon black (15%) show extremely low reversible capacity as well (Fig. 12a and 12b). In this case, carbon additive does not provide sufficient electrical contact between active material grains. Due to this fact, even under low C/200 rate samples performed very poorly in charge/discharge

**Figure 11.** Cell cycling behavior of: a) C/Li2MnSiO4@600, b) C/Li2MnSiO4@700.

**Figure 12.** Cell cycling behavior of standard composites Li2MnSiO4 + carbon black (15% carbon content)

**Figure 13.** Cell cycling behavior of CCL/Li2MnSiO4@600 (7.5% carbon content)

C/Li2MnSiO4 Nanocomposite Cathode Material for Li-Ion Batteries 77

**Figure 16.** Cell cycling behavior of CCL/Li2MnSiO4@700 (10% carbon content)

capacity is limited by carbon coating performance.

**6. Conclusions**

Fig. 13-16 show cycling behavior of CCL composites with different amounts of carbon. Delithation of CCL composites after initial charging is in the range of 48-68% (charged CCL/Li2MnSiO4@600\_12%C – correspond to composition Li1.04MnSiO4 and charged CCL/Li2MnSiO4@700\_10%C – correspond to composition Li0.64MnSiO4). The observed capacity loss in the first cycles is related to SEI formation in CCL. During lithium extraction, Mn3+ and Mn4+ appear, and at the same time Li2MnSiO4 undergoes decomposition caused by Jahn-Teller distortion associated with changes in lattice parameters during Mn3+ → Mn4+ transition [30]. Despite the fact that crystalline structure collapses, discharge capacity is around 90-110 mAh/g. CCL composites produced from Li2MnSiO4 calcined at 600 °C exhibit better coulombic efficiency than Li2MnSiO4 calcined at 700 °C, the reversible capacity after 10 cycle is close to 100 mAh/g. The results indicate that coulombic efficiency of cathode material depends on grain size and homogeneity in grain size distribution, while cell

Dilithium manganese orthosilicate – a high energy density cathode material – was successfully synthesized by sol-gel Pechini method. Encapsulation of nanosized grains of Li2MnSiO4 in carbon matrix, resulted from organic precursor, avoided further sintering. Different crystallite size was obtained, in particular, nanoparticles within range of 5-10 nm. High chemical stability of this material under a highly reductive environment was observed. Application of carbon coating improved electrical conductivity of cathode material to the

**Figure 14.** Cell cycling behavior of CCL/Li2MnSiO4@600 (12% carbon content)

**Figure 15.** Cell cycling behavior of CCL/Li2MnSiO4@700 (5% carbon content)

**Figure 16.** Cell cycling behavior of CCL/Li2MnSiO4@700 (10% carbon content)

Fig. 13-16 show cycling behavior of CCL composites with different amounts of carbon. Delithation of CCL composites after initial charging is in the range of 48-68% (charged CCL/Li2MnSiO4@600\_12%C – correspond to composition Li1.04MnSiO4 and charged CCL/Li2MnSiO4@700\_10%C – correspond to composition Li0.64MnSiO4). The observed capacity loss in the first cycles is related to SEI formation in CCL. During lithium extraction, Mn3+ and Mn4+ appear, and at the same time Li2MnSiO4 undergoes decomposition caused by Jahn-Teller distortion associated with changes in lattice parameters during Mn3+ → Mn4+ transition [30]. Despite the fact that crystalline structure collapses, discharge capacity is around 90-110 mAh/g. CCL composites produced from Li2MnSiO4 calcined at 600 °C exhibit better coulombic efficiency than Li2MnSiO4 calcined at 700 °C, the reversible capacity after 10 cycle is close to 100 mAh/g. The results indicate that coulombic efficiency of cathode material depends on grain size and homogeneity in grain size distribution, while cell capacity is limited by carbon coating performance.

#### **6. Conclusions**

76 Composites and Their Properties

**Figure 14.** Cell cycling behavior of CCL/Li2MnSiO4@600 (12% carbon content)

**Figure 15.** Cell cycling behavior of CCL/Li2MnSiO4@700 (5% carbon content)

Dilithium manganese orthosilicate – a high energy density cathode material – was successfully synthesized by sol-gel Pechini method. Encapsulation of nanosized grains of Li2MnSiO4 in carbon matrix, resulted from organic precursor, avoided further sintering. Different crystallite size was obtained, in particular, nanoparticles within range of 5-10 nm. High chemical stability of this material under a highly reductive environment was observed. Application of carbon coating improved electrical conductivity of cathode material to the satisfactory level of ~10-0.5 S/cm. Thickness of carbon coating on active material grains strongly limits the electrochemical performance of composite. Formation of CCL/Li2MnSiO4 composites significantly improved electrochemical performance of cathode materials, showing a reversible capacity of 90-110 mAh/g after 10 cycles. Electrochemical tests indicated that composite preparation should be optimized in terms of carbon content, CCL performance and homogeneity of a crystallite size. It was found that coulombic efficiency of cathode material depends on grain size and homogeneity in grain size distribution, while cell capacity is limited by carbon coating performance.

C/Li2MnSiO4 Nanocomposite Cathode Material for Li-Ion Batteries 79

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## **Author details**

Marcin Molenda\* , Michał Świętosławski and Roman Dziembaj *Jagiellonian University, Faculty of Chemistry, Krakow, Poland*

## **Acknowledgement**

This work has been financially supported by the Polish National Science Centre under research grant no. N N209 088638, and by the European Institute of Innovation and Technology under the KIC InnoEnergy NewMat project. The part of the measurements was carried out with the equipment purchased thanks to the financial support of the European Regional Development Fund in the framework of the Polish Innovation Economy Operational Program (POIG.02.01.00-12-023/08). One of the authors (M.Ś.) acknowledge a financial support from the International PhD-studies programme at the Faculty of Chemistry Jagiellonian University within the Foundation for Polish Science MPD. TEM analysis were carried out in Laboratory of Transmission Analytical Electron Microscopy at the Institute of Metallurgy and Material Science, Polish Academy of Sciences.

## **7. References**

<sup>\*</sup> Corresponding Author

[6] Molenda M., Dziembaj R., Podstawka E., Proniewicz L.M. Changes in local structure of lithium manganese spinels (Li:Mn=1:2) characterised by XRD, DSC, TGA, IR, and Raman spectroscopy. Journal of Physics and Chemistry of Solids 2005;66(10) 1761-1768.

78 Composites and Their Properties

**Author details**

Marcin Molenda\*

Sciences.

 \*

Corresponding Author

**7. References**

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**Acknowledgement**

cell capacity is limited by carbon coating performance.

*Jagiellonian University, Faculty of Chemistry, Krakow, Poland*

satisfactory level of ~10-0.5 S/cm. Thickness of carbon coating on active material grains strongly limits the electrochemical performance of composite. Formation of CCL/Li2MnSiO4 composites significantly improved electrochemical performance of cathode materials, showing a reversible capacity of 90-110 mAh/g after 10 cycles. Electrochemical tests indicated that composite preparation should be optimized in terms of carbon content, CCL performance and homogeneity of a crystallite size. It was found that coulombic efficiency of cathode material depends on grain size and homogeneity in grain size distribution, while

, Michał Świętosławski and Roman Dziembaj

This work has been financially supported by the Polish National Science Centre under research grant no. N N209 088638, and by the European Institute of Innovation and Technology under the KIC InnoEnergy NewMat project. The part of the measurements was carried out with the equipment purchased thanks to the financial support of the European Regional Development Fund in the framework of the Polish Innovation Economy Operational Program (POIG.02.01.00-12-023/08). One of the authors (M.Ś.) acknowledge a financial support from the International PhD-studies programme at the Faculty of Chemistry Jagiellonian University within the Foundation for Polish Science MPD. TEM analysis were carried out in Laboratory of Transmission Analytical Electron Microscopy at the Institute of Metallurgy and Material Science, Polish Academy of

[1] Whittingham M.S. Electrical Energy Storage and Intercalation Chemistry. Science

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[3] Tarascon J.-M., Guyomard D. The Li1+xMn2O4/C rocking-chair system: a review

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**Section 2**

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**Theoretical and Numerical Modeling**

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[24] Mali G., Rangusa M., Sirisopanaporn C., Dominko R. Understending 6Li MAS NMR spectra of Li2MSiO4 materials (M=Mn, Fe, Zn). Solid State Nuclear Magnetic Resonance

[25] Liu W., Xu Y., Yang R. Synthesis, characterization and electrochemical performance of Li2MnSiO4/C cathode material by solid-state reaction. Journal of Alloys and

[26] Karthikeyan K., Aravindan V., Lee S.B., Jang I.C., Lim H.H., Park G.J., Yoshio M., Lee Y.S. Electrochemical performance of carbon-coated lithium manganese silicate for asymmetric hybrid supercapacitors. Journal of Power Sources 2010;195(11) 3761-3764. [27] Gummow R.J., Sharma N., Peterson V.K., He Y. Crystal chemistry of the *Pmnb*

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**Chapter 5**

© 2012 Escárpita et al., licensee InTech. This is an open access chapter 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.

© 2012 Escárpita et al., licensee InTech. This is a paper 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.

**Biaxial Tensile Strength Characterization**

David Alejandro Arellano Escárpita, Diego Cárdenas, Hugo Elizalde,

Woven architecture confers textile composites (TC) multidirectional reinforcement while the undulating nature of fibres also provides a certain degree of out-of-plane reinforcement and good impact absorption; furthermore, fibre entanglement provides cohesion to the fabric and makes mould placement an easy task, which is advantageous for reducing production times [1]. These features make TC an attractive alternative for the manufacture of highperformance, lightweight structural components. Another interesting feature of TC is that they can be entangled on a variety of patterns, depending of the specific applications intended. Despite the wide interest of textile composites for industry and structural applications, most of the research efforts for strength characterization has focused on unidirectional composites (UDC), resulting in a large number of failure theories developed for UDC (around 20, as inferred from the conclusions of the World-Wide Failure Excercise (WWFE) [2]); some of the most popular failure models are used indistinctly for UDC and TC: most designers use Maximum Strain, Maximum Stress, Tsai-Hill and Tsai-Wu both for UDC or TC as stated in reference [3], despite the fact that none of the aforementioned failure criteria has been developed specifically for TC, which has led to use of high safety factors in critical structural applications to overcome associated uncertainties [4]. The most successful approaches to predict TC strength are based on phenomenological modeling of interactions between constituents at different scales (matrix-yarn-fiber), providing new insight into TC failure mechanisms. However, the implementation of phenomenological models as design tools is considerably more complex than that of traditional failure criteria, while still exhibiting significant deviation from the scarce experimental data [5] available. This scarcity of experimental data to validate or reject failure theories continues to be a major obstacle for improving TC models. Recent investigations reporting biaxial tensile strength tests in 2D-

**of Textile Composite Materials**

Ricardo Ramirez and Oliver Probst

http://dx.doi.org/10.5772/48105

**1. Introduction**

Additional information is available at the end of the chapter

**Chapter 5**


**3.4 LYVE-1**

HA links the two main functions of the lymphatic system: draining of interstitial fluids and immune surveillance. These functions are achieved through its interaction with the receptor LYVE-1, present in lymphatic endothelia (Jackson, 2009). LYVE-1 is a type I integral membrane polypeptide that exhibits high homology with CD44 (Banerji *et al*., 1999) and is a homeostatic HA receptor required for liver and lymphatic vessel formation. Its expression does not change as frequently in malignancy as HA receptors involved in response to injury, for example CD44 and RHAMM/HMMR. This does not rule out a role in injury and tumour progression however, as lymphangiogenesis is an important processes in both events, and elevated accumulation of HA in stroma results in lymphangiogenesis *via* signalling through LYVE-1 (Gale *et al*., 2007).

To further demonstrate the association of LYVE-1 with tumour dissemination through the lymphatic system, (Du *et al*., 2010) expressed LYVE-1 in COS-7 kidney cells and performed cell adhesion assays with the BCA cell line HS-578T which produces HA. These two cell lines had enhanced adhesion over the control cells, COS-7 not expressing LYVE-1. This

tumour cell migration/infiltration. The human BCA cell line MDA-MB-231 expresses mainly TLR4, and siRNA mediated knock-down of TLR4 significantly reduces cell survival and expression of the cytokines Il-6 and Il-8, suggesting that TLR4 is a promising target for

Fig. 3. HA initiates the signalling of RHAMM and CD44 regulated pathways, resulting in a

HA links the two main functions of the lymphatic system: draining of interstitial fluids and immune surveillance. These functions are achieved through its interaction with the receptor LYVE-1, present in lymphatic endothelia (Jackson, 2009). LYVE-1 is a type I integral membrane polypeptide that exhibits high homology with CD44 (Banerji *et al*., 1999) and is a homeostatic HA receptor required for liver and lymphatic vessel formation. Its expression does not change as frequently in malignancy as HA receptors involved in response to injury, for example CD44 and RHAMM/HMMR. This does not rule out a role in injury and tumour progression however, as lymphangiogenesis is an important processes in both events, and elevated accumulation of HA in stroma results in lymphangiogenesis *via*

To further demonstrate the association of LYVE-1 with tumour dissemination through the lymphatic system, (Du *et al*., 2010) expressed LYVE-1 in COS-7 kidney cells and performed cell adhesion assays with the BCA cell line HS-578T which produces HA. These two cell lines had enhanced adhesion over the control cells, COS-7 not expressing LYVE-1. This

BCA therapy (Yang *et al.*, 2010).

variety of pro-tumourigenic outcomes.

signalling through LYVE-1 (Gale *et al*., 2007).

**3.4 LYVE-1**

suggests that LYVE-1 plays a role in tumour cell adhesion which is dependent on HA-LYVE-1 interaction. Apart from its effect on tumour cell adhesion, LYVE-1 has also been proven to be a prognostic factor in tongue squamous cell carcinoma and decreased levels of LYVE-1 in the invasive front of tumours predicts cervical lymph node metastasis (Matsumoto *et al*., 2010).


**5.1. Advantages**

**1.** Limited Constraints: Being air borne they are not hindered by physical constraints such as road/soil terrain, uneven paths and obstacles. They can simply fly over them all.


*Article* **A Comparative Analysis of Methods (LC-MS/MS, LC-MS and Rapid Test Kits) for the Determination of Diarrhetic Shellfish Toxins in Oysters, Mussels and Pipis**

**Penelope A. Ajani 1,\* , Chowdhury Sarowar 2 , Alison Turnbull 3 , Hazel Farrell 4 , Anthony Zammit 4 , Stuart Helleren 5 , Gustaaf Hallegraeff 3 and Shauna A. Murray 1**

**Abstract:** Rapid methods for the detection of biotoxins in shellfish can assist the seafood industry and safeguard public health. Diarrhetic Shellfish Toxins (DSTs) are produced by species of the dinoflagellate genus *Dinophysis*, yet the comparative efficacy of their detection methods has not been systematically determined. Here, we examined DSTs in spiked and naturally contaminated shellfish– Sydney Rock Oysters (*Saccostrea glomerata*), Pacific Oysters (*Magallana gigas*/*Crassostrea gigas*), Blue Mussels (*Mytilus galloprovincialis*) and Pipis (*Plebidonax deltoides*/*Donax deltoides*), using LC-MS/MS and LC-MS in 4 laboratories, and 5 rapid test kits (quantitative Enzyme-Linked Immunosorbent Assay (ELISA) and Protein Phosphatase Inhibition Assay (PP2A), and qualitative Lateral Flow Assay (LFA)). We found all toxins in all species could be recovered by all laboratories using LC-MS/MS (Liquid Chromatography—tandem Mass Spectrometry) and LC-MS (Liquid Chromatography—Mass Spectrometry); however, DST recovery at low and mid-level concentrations (<0.1 mg/kg) was variable (0–150%), while recovery at high-level concentrations (>0.86 mg/kg) was higher (60–262%). While no clear differences were observed between shellfish, all kits delivered an unacceptably high level (25–100%) of falsely compliant results for spiked samples. The LFA and the PP2A kits performed satisfactorily for naturally contaminated pipis (0%, 5% falsely compliant, respectively). There were correlations between spiked DSTs and quantitative methods was highest for LC-MS (r2 = 0.86) and the PP2A kit (r2 = 0.72). Overall, our results do not support the use of any DST rapid test kit as a stand-alone quality assurance measure at this time.

**Keywords:** LC-MS; rapid test kit; biotoxins; shellfish; diarrhetic shellfish toxins; *Dinophysis*

**Key Contribution:** LC-MS continues to be a reliable DST detection method across labs and shellfish species; with low to mid-level toxin concentration recovery more variable than high-level. All rapid test kits delivered unacceptably high, falsely compliant results for spiked samples. The Neogen and the PP2A kits performed satisfactorily for naturally contaminated pipis. Overall, our results do not support the use of any DST rapid test kit as a stand-alone quality assurance measure at this time.

## **1. Introduction**

Marine biotoxins are toxic chemical compounds produced by certain microalgae, which can bioaccumulate in shellfish and other marine organisms, and cause poisoning to

**Citation:** Ajani, P.A.; Sarowar, C.; Turnbull, A.; Farrell, H.; Zammit, A.; Helleren, S.; Hallegraeff, G.; Murray, S.A. A Comparative Analysis of Methods (LC-MS/MS, LC-MS and Rapid Test Kits) for the Determination of Diarrhetic Shellfish Toxins in Oysters, Mussels and Pipis. *Toxins* **2021**, *13*, 563. https://doi.org/ 10.3390/toxins13080563

Received: 8 July 2021 Accepted: 9 August 2021 Published: 11 August 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 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 (https:// creativecommons.org/licenses/by/ 4.0/).

seafood consumers. As well as seafood related illnesses, marine biotoxin contamination can lead to damaged public perceptions of seafood, direct economic losses and a restriction in the growth of the shellfish industry.

Diarrhetic Shellfish Toxins (DSTs) are produced by dinoflagellates of the planktonic genus *Dinophysis* and *Phalacroma*, and more rarely benthic *Prorocentrum*, and can bioaccumulate in shellfish and cause Diarrhetic Shellfish Poisoning (DSP). With approximately 11,000 human poisonings reported globally over the period 1985–2018 [1], DSP is a gastrointestinal disorder caused by the human consumption of seafood contaminated with DSTs. While symptoms are dose dependent and include diarrhea, nausea, vomiting and abdominal pain, DSTs are potent inhibitors of certain protein phosphatases and may promote tumor/cancer formation [2], although the impact of chronic exposure to DSTs is still not well known.

DSTs are a group of heat stable, polyether toxins consisting of okadaic acid (OA) and its isomer 19-epi-okadaic acid; the OA congeners dinophysistoxin-1 (DTX-1) and dinophysistoxin-2 (DTX-2); and the 7-acyl derivatives of OA, DTX-1 and DTX-2 that are collectively known as DTX-3. Together, they are referred to as the OA group toxins or the 'okadaates' (OAs). While OA, DTX-1 and DTX-2 only differ slightly in their molecular structure, the DTX-3 (group) includes a wide range of derivatives esterified with saturated and unsaturated fatty acids, products of metabolic transformations that occur in the shellfish [3]. Chemical compounds of this group are therefore generally described as either 'free' (unesterified) or 'esterified' [4].

DSP was first described after a large toxin event occurred in Japan in 1976 [5,6], whereby many people became sick after eating scallops (*Patinopecten yessoensis*). This contamination was linked to toxins produced by *Dinophysis fortii*. Following this event, further toxic episodes occurred in Japan, Spain and France, with several thousands of cases of human poisonings occurring over the 1970s and 1980s, and leading to the development of many regional monitoring programs. This monitoring has seen a gradual increase in reported DSP episodes in countries including Chile, Argentina, Mexico, the east coast of North America, Scandinavia, Ireland, Great Britain, Spain, Portugal, Italy, Greece, India, Thailand, Australia and New Zealand [5,7–9].

*Dinophysis* is common in Australian waters, with 36 species reported [10–12]. Toxic species include *D. acuminata* Claparede and Lachmann, *D. acuta* Ehrenberg, *D. caudata* Saville-Kent, *D. fortii* Pavillard, *D. norvegica* Claparede and Lachmann and *D. tripos* Gourret. There have been three serious DSP events in Australia. The first episode was caused by contamination of Pipis (*Plebidonax deltoides*) in New South Wales in 1997 (NSW) by *D. acuminata* [13]. One hundred and two people were affected and 56 cases of gastroenteritis were reported. A second episode occurred again in NSW in March 1998, this time with 20 cases of DSP poisoning reported [14]. The final event occurred in Queensland in March 2000, which was again linked to the consumption of Pipis [15]. While no human fatalities from DSP are known globally, DSTs continue to be a major food safety challenge for the shellfish industry.

Detection methods for DSTs using liquid chromatography with tandem mass spectrometry (LC-MS/MS and LC-MS) [4,16] and implemented as part of seafood safety programs, are considered the "gold standard" across the globe. These methods replaced the mouse bioassay (MBA), which was previously the most commonly used laboratory analysis tool (e.g., [17]). However, the development of more rapid, cost effective (on farm) testing methods for the presence of DSTs would potentially make harvest management simpler and faster and result in fewer closures. Three types of rapid test kits for the detection of DSTs are currently commercially available. These include an antibody-based enzyme-linked immunosorbent assay (ELISA) test; a functional protein phosphatase inhibition activity (PPIA) assay; and a lateral flow analysis (LFA) rapid test. ELISA assays involve an antigen immobilized on a (micro) plate, which are then complexed with an antibody that is linked to a reporter enzyme. These assays were first developed in the 1960s and 1970s for primarily medical diagnosis purposes [18]. Detection of OA, DTX-1 and DTX-2 (varying analogue

cross reactivity depending on kit) is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a quantifiable product. Functional PPIA assays quantify okadaic acid (OA) and DST analogues including DTX-1, DTX-2 and DTX3 by colorimetric phosphatase inhibition, based on the reversible inhibition of protein phosphatase type 2A (PP2A) by the toxin, and the resulting absorbance derived from enzymatic hydrolysis of the substrate. A lateral flow test involves the shellfish extract transported across a reagent zone in which OA, DTX-1, DTX-2 and DTX-3 specific antibodies are combined with colored particles. If a toxin is present, it is captured by the particle-antibody complex, and as its concentration increases, the intensity of the test "line" decreases [19].

In a comprehensive review by McLeod et al. [20] of the currently available field methods for detection of marine biotoxins in shellfish, it was concluded that the ELISAs and LFAs had poor reactivity to the DSP congener DTX-2 and can give false negative results when high levels of DTX-3 are present (and the hydrolysis step is not undertaken to release ester forms). LFAs were also found to give some false positive results when DSP was below the ML (Max Limit), but this was dependent on the toxin profile, geographic region and shellfish species involved. Pectenotoxins (PTXs) are not currently included in Codex Standard for Live and Raw Bivalve Molluscs [21], and therefore are not included in this study. Several other jurisdictions such as Canada, Chile and the European Union do regulate for PTX (but not PTX-2sa), but the European Food Safety Authority has issued an opinion to deregulate PTX [22]. Furthermore, DSP regulation in Australia is governed by Food Standards Australia New Zealand with a maximum regulatory limit of 0.2 mg OA eq/kg [23], while most international standards including the Codex Standard, state a ML of 0.16 mg OA eq/kg [21].

To date, these rapid detection kits have not been tested on various shellfish matrices in a systematic manner, nor a comparison made across multiple analytical laboratories to assess LC-MS/MS or LC-MS detection of DSTs in shellfish. With this in mind, the present study aimed to undertake a comparative study to detect DSTs in differing shellfish matrices using commonly implemented protocols for LC-MS/MS or LC-MS in several different laboratories, as well as compare five commercially available rapid test kits for the detection of DSTs in these same shellfish tissues. The rapid test kits included three quantitative ELISA kits by BeaconTM, Eurofins/AbraxisTM and EuroProximaTM; a quantitative PP2A kit by Eurofins/AbraxisTM, and a qualitative LFA kit by NeogenTM.

#### **2. Results**

#### *2.1. LC-MS/MS and LC-MS*

No toxins were detected in any of the four shellfish species matrices (Sydney Rock Oysters (*Saccostrea glomerata*) (SRO), Pacific Oysters (*Magallana gigas*/*Crassostrea gigas*) (PO), Blue Mussels (*Mytilus galloprovincialis*) (MUS) and Pipis (*Plebidonax deltoides*/*Donax deltoides*) (PIPI)) screened before spiking began (see Methods). Of the triplicate SROs spiked with OA at 0.02 mg/kg, Laboratory 1 detected OA in all three samples (x = 0.01, SD ± 0.00, min <0.01, max 0.02 mg/kg), Laboratory 2 and 4 reported concentrations below the detection limit for all samples (<0.01 mg/kg and <0.025 mg/kg respectively), and Laboratory 3 detected OA in all three samples (x = 0.013, SD ± 0.006, min 0.01, max 0.02 mg/kg). In summary, two out of the four laboratories detected OA at this low level, with recoveries between ~50–100% (Table 1).

Of the four shellfish species spiked with OA at 0.02 mg/kg, Laboratory 1 detected this toxin in all four matrices (x = 0.013, SD ± 0.005, min 0.01, max 0.02 mg/kg), Laboratory 2 did not detect OA in SRO or PO; however, it was detected in both MUS and PIPI (x = 0.015, SD ± 0.007; min <0.01, max 0.02 mg/kg), and Laboratory 3 did not detect OA in PO or MUS, but detected it in SRO and PIPI (x = 0.015, SD ± 0.007; min <0.01, max 0.02 mg/kg). Laboratory 4 did not detect OA at this concentration (less than detection limit <0.025 mg/kg). Laboratory 4, however, did detect OA in one PIPI sample at 0.03 mg/kg (>spike concentration). In summary, OA was detected in all matrices at this concentration, although not all laboratories detected toxins in all four matrices. Recovery across all laboratories ranged from ~50–150% (Table 2).

**Table 1.** Results of LC-MS/MS (Liquid Chromatography—tandem Mass Spectrometry) and LC-MS (Liquid Chroma-tography—Mass Spectrometry) for Sydney Rock Oysters (SRO) spiked with 0.02 mg/kg okadaic acid (no DTX-1 or DTX-2 added).

<LOR = below limit of reporting; Note: Spike below limit of reporting for Laboratory 4.

**Table 2.** Results of LC-MS/MS and LC-MS for Australian shellfish—Sydney Rock Oysters (SRO), Pacific Oysters (PO), Blue Mussels (MUS) and Pipis (PIPI) spiked with 0.02 mg/kg okadaic acid (no DTX-1 or DTX-2 added).

<LOR = below limit of reporting; Note: Spike below limit of reporting for Laboratory 4.

For the shellfish spiked with DTX-1 at 0.04 mg/kg, Laboratory 1 recovered this analogue in all matrices (x = 0.035, SD ± 0.006; min 0.03, max 0.05 mg/kg), with one PIPI sample returning a concentration of 0.01 OA mg/kg. Laboratory 2 detected DTX-1 in all matrices (x = 0.025, SD ± 0.006; min 0.02, max 0.03 mg/kg), also with a detection of OA in PIPI at 0.02 mg/kg. Laboratory 3 detected DTX-1 in all matrices (x = 0.025, SD ± 0.006; min 0.02, max 0.03 mg/kg), while Laboratory 4 did not detected this toxin in MUS (other matrices x = 0.026, min <0.025, max 0.04 mg/kg) (Table 3). In summary, DTX-1 was detected in all shellfish matrices at this concentration; however, one laboratory did not detect DTX-1 in MUS. The overall recovery of this analogue was ~50–100% across laboratories with two detections of OA in PIPIs.

For all shellfish spiked with DTX-2 at 0.01 mg/kg, Laboratory 1 did not recover this analogue in SRO or PIPI, and was only detected it in PO and MUS (both at 0.01 mg/kg). No toxin at this concentration was recovered from either Laboratory 2 nor Laboratory 3, while Laboratory 4 was unable to detect this toxin (below the limit of reporting <0.025 mg/kg) (Table 4). In summary DTX-2 was only detected in PO and MUS at this low concentration, and only at one laboratory. Overall recovery was ~50–100%.

**Table 3.** Results of LC-MS/MS and LC-MS for Australian shellfish—Sydney Rock Oysters (SRO), Pacific Oysters (PO), Blue Mussels (MUS) and Pipis (PIPI) spiked with 0.04 mg/kg DTX-1 (no OA or DTX-2 added).

<LOR = below limit of reporting.

**Table 4.** Results of LC-MS/MS and LC-MS for Australian shellfish—Sydney Rock Oysters (SRO), Pacific Oysters (PO), Blue Mussels (MUS) and Pipis (PIPI) spiked with 0.01 mg/kg DTX-2 (no OA or DTX-1 added).

<LOR = below limit of reporting; Note: Spike below limit of reporting for Laboratory 4.

When shellfish were spiked with all toxins (in varying concentrations between 2–10 × LOR depending on toxin analogue; see Methods), laboratory recovery of total toxin per sample for each laboratory was as follows: Laboratory 1: 53–75%; Laboratory 2: 35–88%; Laboratory 3: 13–41%; and Laboratory 4: 0–88% (Table 5). More specifically, all toxins were recovered in all matrices for Laboratory 1, with an individual toxin/sample recovery ranging from 40–200%, with the lowest matrix average recovery in SRO at 57% and the highest in PIPI at 103%. For Laboratory 2, DTX-2 was not detected in SRO or PO, while individual toxin/sample recovery ranged from 40–400%, with the lowest matrix average recovery in SRO at 43%, and the highest in PIPI at 170%. For Laboratory 3, OA was not detected in MUS or PIPI, and DTX-2 was not detected in PIPI. The individual toxin/sample recovery ranged from 20–50%, with the lowest matrix average in PIPI at 40% and the highest in MUS at 47%. Finally, for Laboratory 4, DTX-2 was not detected across all matrices and OA was not detected in MUS. Individual toxin/sample recovery ranged from 50–340% with the lowest matrix average in MUS at 50% and the highest in PIPI at 154%. Overall, most toxins were detected by all laboratories at these concentrations, individual recovery across all labs/matrices ranged from 0–88%, while the recovery across shellfish matrices varied.

In our final analysis to determine the recovery of CRM (OA/DTX-1/DTX-2), all laboratories detected all toxin analogues. Individual toxin recoveries ranged from 88 to 131% for Laboratory 1, 79–81% for Laboratory 2, 83–95% for Laboratory 3 and 101–262% for Laboratory 4 (Table 6). However, considering that these recoveries are the result of one sample per lab, they should be treated as indicative only.

**Table 5.** Results of LC-MS/MS and LC-MS for Australian shellfish—Sydney Rock Oysters (SRO), Pacific Oysters (PO), Blue Mussels (MUS) and Pipis (PIPI) spiked with a combination of DST analogues-OA 0.1 mg/kg; DTX-1 0.05 mg/kg; and DTX-2 0.02 mg/kg.

<LOR = below limit of reporting; Note: Spike of OA for MUS and PIPI below limit of reporting for Laboratory 4.

**Table 6.** Results of LC-MS/MS and LC-MS for Certified Reference Material CRM DSP-Mus-c.

\* CRM are certified for free toxin; they report higher total toxin concentration post hydrolysis but these are not certified.

#### *2.2. Rapid Test Kits*

#### 2.2.1. Wild Harvest Pipis

Prior to rapid test kit screening, OA, DTX-1 and DTX-2 analysis by LC-MS for wild harvest Pipis resulted in a OA toxin range of 0.1 to 0.3 mg/kg (Sample 4A—0.1 mg/kg, 4B—0.1 mg/kg, 4C—0.2 mg/kg, and 4D—0.3 mg/kg). After hydrolysis, no DTX-1 or DTX-2 was detected in any samples. Three batches comprising 10 replicates of each OA toxin concentration of 0.1, 0.2 and 0.3 mg/kg were subsequently screened using each rapid test kit.

#### 2.2.2. LC-MS

Using LC-MS (Laboratory 3), all control shellfish samples (no toxin added) returned a 'not detected' result (Table 7). For OA spiked samples, 43/46 (~93%) returned concentrations at, or slightly above, the spiked toxin concentrations 0.1 and 0.2 mg/kg (Tables 7 and 8). The three samples (7%) that returned concentrations lower that the spiked

concentration were all spiked Pipi samples: sample 22 reported 0.09 mg/kg when it was spiked with OA at 0.1 mg/kg; sample 23 reported 0.15 mg/kg when it was spiked with OA at 0.2 mg/kg; and finally, sample 24 reported 0.09 mg/kg when it was spiked with OA at 0.2 mg/kg (Tables 7 and 8). The latter two of these samples were falsely compliant at the regulatory limit (7%, 2/28). A Pearson's correlation analysis between LC-MS results and the concentration of spiked toxin revealed a very strong relationship (r2 = 0.86) (Figure 1). Subsequently, this method returned a mean recovery of 106.5%, meeting the criteria set out in the AOAC Guidelines for Single Laboratory Validation of Chemical Methods for Dietary Supplements and Botanicals (AOAC 2002).

**Table 7.** Results of LC-MS and rapid test kits for Okadaic Acid spiked into Australian shellfish (Sydney Rock Oysters [SRO], Pacific Oyster [PO], Blue Mussel [MUS] and Pipis [PIPI]). Note: Neogen qualitative test (±) with Limit of Quantification = 0.08 mg/kg; Abraxis PP2A Working Range = 0.06 to 0.35 mg/kg; Beacon ELISA Limit of Quantification = 0.1 mg/kg; Abraxis ELISA Working Range = 0.1–5.0 mg/kg; Europroxima ELISA Limit of Quantification = 0.04 mg/kg.

ND = not detected (0.01 mg/kg detection limit).

2.2.3. Rapid Test Kits

Qualitative Test

Neogen

The Neogen kit returned negative readings for the eight negative control samples across all species-specific shellfish matrices. However, 23 out of 46 samples (50%) of spiked samples (across all shellfish matrices) returned a negative result when they contained okadaic acid (Tables 7 and 8). Within this group, 18% (5/28 samples again across all matrices) returned a false compliant result when they were spiked at, or above, the regulatory limit (=/> 0.2 mg OA eq/kg), while no naturally contaminated Pipis returned falsely compliant results with this kit.