Croatian diatomites and their possible application as a natural insecticide

In recent decades, there has been an increase in the use of diatomaceous earth (DE) as a natural insecticide because of its low mammalian toxicity, worker safety, low risk of food residues and the occurrence of resistant insect populations associated with the use of chemical insecticides. Therefore there is potential for research into known but previously undescribed Croatian midMiocene marine diatomites from the perspective of their potential as proper DE that could be mixed with plant extracts as a new formulation for grain storage protection. The marine diatomites belong to the Paratethyan near shore environment, deposited in the upwelling zone during a mid-Miocene temperate climate. Palaeontological, mineral and geochemical analyses were done on ten promising marly sediments from 26 outcrops and one borehole from the North Croatian Basin. The most important ingredient of diatomaceous sediments is silica (biogenic opal-A and SiO2 bound in other silicate minerals including quartz, clay minerals, micas, etc.). The amorphous silica content of the tested Croatian diatomites is relatively low ( 50%) in comparison with the Celatom® MN 51 standard (medium to high efficient DE) (73.6%), nevertheless they show in some part even slightly better efficacy against insects. It seems that the enhanced content of smectite in diatomaceous sediments also influences increased absorption of DE. Based on palaeontological results, the most efficient diatomites from the Podsusedsko Dolje and Markuševec (Medvednica Mt.) consist of the mid-sized planktonic Coscinodiscus group of species where Thalassionema nitzschioides dominate and is positively correlated with their absorption. The usage of Boströms’ standard formula for getting opal-A from geochemical data was abandoned because of negative results and the modified Murdmaas’ formula for hemipelagic sediments was applied. Preliminary results on the aforementioned diatomite (as inert dusts) show good efficacy against tested insects Sitophilus oryzae (LINNAEUS), Tribolium castaneum (HERBST) and Rhyzopertha dominica (FABRICIUS). Article history: Manuscript received November 17, 2016 Revised manuscript accepted February 24, 2017 Available online February 28, 2017


INTRODUCTION
Diatomite is a porous, lightweight, siliceous sedimentary rock mainly composed of diatom skeletons formed in marine to freshwater environments of mid to high latitudes and contains >30% amorphous opaline silica (SiO 2 •nH 2 O).Commercial diatomite contains >60% of biogenic silica (BSi).Mostly high-grade diatomite (>80% BSi) is used as diatomaceous earth (DE), crushed and oven-dried inert dust with a minor content of calcium carbonate, volcanic glass and terrigenous particles (HARWOOD, 2010).Sediments that contains <30% of diatoms belongs to the tripolite.
Biogenic silica is non toxic to mammals and has been registered in many countries as a food additive.DE is known as a potentially useful grain protectant because it is safe to use, does not affect grain end-use quality, provides long-term protection and is comparable in cost to other methods of grain protection (KORUNIĆ, 2013).Ideally, active DE should have a high BSi content with a uniform particle size (less than 10 microns), a high oil sorption capacity, a large active surface, and very little clay and other impurities (KORUNIĆ, 1998).Lately, there are nume rous reports from around the world on the widespread resistance of several stored-product insects to grain protectants from the groups of synthetic pyrethroids, organophosphates, carbonates, chlorinated hydrocarbons, Bacilus thuringiensis, botanicals and chemicals (SUBRAMANYAM & HAGSTRUM, 1995;KLJAJIĆ & PERIĆ, 2005).Since only a physical method of controlling insects is involved in DE, genetic resistance is generally unlikely according to EBELING (1971).However, a potential tolerance or even resistance should be considered when choosing DE for replacing existing grain protectants (KORUNIĆ, 1998;RIGAUX et al., 2001;FIELDS, 2003;VAYIAS et al., 2008).
The efficacy of DE against insects depends on the different properties of the diatom particles, including physical and morphological characteristics of diatoms, rather than on its origin.The physical property of diatomite is in its specific gravity (density) of diatom frustules (skeletons), which is about half that of water.The perforations (pores and striae) and open structure of diatom frustules renders diatomite a considerably lower density (0.12-0.25 g/cm 3 ) and high porosity (75-85%), able to absorb and hold up to 3.5 times its own weight in liquid i.e. in this case wax.Well known diatoms with pores (areolas) have the physical ability to absorb wax (lipids) from the insects' epicuticle, causing its dehydration and death (EBELING, 1971;KORUNIĆ, 1998), according to high absorbency which speeds up the incorporation of oil.
The earliest known Croatian diatomites are from the Podsusedsko Dolje outcrops (Medvednica Mt.), and can be described as diatomaceous tripolite (outcrops J1J4: PANTOCSEK, 1903 System (PBS) (Central Paratethys) (PAVELIĆ, 2001;PAVE LIĆ et. al., 2003) (Fig. 1).This intracontinental region began to develop in the early Miocene as a result of the African and European plate collision (HORVÁTH & ROYDEN, 1981).PBS is surrounded by the Alps, Dinarides and Carpathian Mountains.The evolution of the Central Paratethys was controlled by regional tectonic events, sea-level changes and connections with the Mediterranean, Boreal and IndoPacific Oceans (STEININGER et al., 1988;RÖGL, 1996).These connections have been repeatedly disrupted and re-established until the end of the Badenian when the sea level dropped, causing the beginning of the isolation and disintegration of the Paratethys.At the beginning of the Sarmatian, the connection with the other marine areas further weakened, and was followed by isolation; a stronger terrestrial influence favoured further decrease in salinity, resulting in changes to flora and fauna and their endemism.This was all more strongly influenced by climate changes.From the warmer period in the Bade nian, the climate during the Sarmatian becomes more moderate with seasonal oscillations (GALOVIĆ, 2001).In the Sarmatian, the connections with the Mediterranean and IndoPacific realms also existed, as shown by the last Miocene transgression (RÖGL, 1998;KOVÁČ et al., 2001), and corroborated by investigations of siliceous microplankton (JURILJ, 1957;HAJÓS, 1986;GALO VIĆ & BAJRAKTAREVIĆ, 2006).The total thickness of the Sarmatian deposits is relatively small (50 to 150 m), they predominantly consist of marly sediments of the tripolite type (BAJRAKTA RE VIĆ et al., 1986) with rare limestones, clastic and volcanic fragments.Such a small thickness is probably a consequence of the basin deepening (due to lithosphere cooling and compression at the end of the Sarmatian) and less sediment input, which is characteristic of the early postrifting phase (PAVELIĆ et al., 2003).1).Tripolite is a broad term, used in Croatia for laminated marly sediments by many of the aforementioned authors.The first applied terminology of diatomite was used by GORJANOVIĆ KRAMBERGER (1908) andJURILJ (1957).During the investigations for the Croatian Basic Geological Map 1:50.000 from 2000 to 2004, a few more outcrops of diatomaceous tripolite (i.e.diatomite) were discovered in Hrvatsko Zagorje (CR), at Medvednica, Žumberak (Jur1 and Jur2) and Požeška gora Mts.Seventy seven more diatom taxa were determined during those investigations on the Podsusedsko Dolje outcrops, where 23 were hitherto not known for the Sarmatian (GALOVIĆ & BAJRAKTAREVIĆ, 2006).
Our investigations are part of the Croatian Science Foundation (HRZZ) project "Development of new natural insecticide formulations based on inert dusts and botanicals to replace synthetic, conventional insecticides" -DIACROMIXPEST conducted from 20142016 (ROZMAN et al., 2015).Throughout this research six more outcrops of diatomite have been discovered in the Banovina region, two in Slavonia and one at Žumberak Mt. (Fig. 1).The palaeontological, mineralogical and geochemical results of Croatian diatomites are presented here with their potential usage as DE in agriculture for the protection of stored products.

GEOLOGICAL SETTING
The investigated mid-Miocene (Sarmatian) sediments of the North Croatian Basin (NCB) and Hrvatsko Zagorje Basin (HZB), belong to the south-western marginal part of the Pannonian Basin  Since the beginning of the Miocene (23 Ma;OGG et al., 2016), the most commercially exploited diatomaceous earth deposits are lacustrine in origin, although the largest producing deposit that outcrops near Lompoc in California is a marine deposit of Miocene age (DOLLEY & MOYLE, 2003).Nevertheless, other diatomites from Paratethys are also marine deposits of Miocene age from Slovenia, Austria, Czech, Slovakia, Hungary and Serbia (ŘEHÁKOVÁ, 1977;HAJÓS, 1986;HORVAT, 2004;ROETZEL et al., 2006;SCHÜTZ et al., 2007;ROJHT et al., 2010).Well known Croatian diatomites from Medvednica Mt. belong to near shore mid-Sarmatian marine sediments characteristic of the upwelling zone (GALOVIĆ & BAJRAKTAREVIĆ, 2006).
Individual parts of the NCB were uplifted and thus became source regions for clastic Sarmatian deposits, accumulating in environments of reduced salinity (VRSALJKO, 1999).Through advanced ingression and deepening of the depositional environment, thin-bedded to laminated marls and sandstones accumulated (VRSALJKO et al., 2005;VRSALJKO, 1997).
In the NCB Sarmatian shallow-water gravels, biocalcarenites, and limestones were laid down, whereas later laminated marly deposits predominated, due to the deepening of the basin.For the region of the Žumberak and Samobor Mts., Sarmatian sediments consisting of calcite-rich marls and limestones with sandstone intercalations, crop out in a narrow zone along the Badenian deposits, (VRSALJKO, 2003).

Palaeontology
The standard preparation method used by the Croatian Geological Survey is applied.Approximately 1 cm 3 of the sediment is placed in a beaker and treated with 30% concentrated hydrogen peroxide (H 2 O 2 ) to oxidize organic matter for light microscope (LM) analyses.Siliceous microfossil slides for SEM preparations also include treatment with 20 % of concentrated HCl acid to remove carbonate and 10 % of sodium pyrophosphate to remove clay components.The sediments are then rinsed with distilled water.Some of the samples were treated in an ultrasonic bath for approximately 15 seconds to improve disaggregation.Standard smear slides and SEM stub preparation techniques were used in slide preparation for both LM and SEM analysis.The specimens were coated with gold and studied with a JEOL JSM35CF SEM.Smear slides were examined using a BH2 Olympus LM.
Composition and diversity of diatom species together with their morphometric analyses are essential for the determination of DE quality.Quantitative methods were undertaken for 300 valves of diatom genera along transects under 500 x magnification (VILIČIĆ, 2003;HORVAT, 2004).Only whole diatom valves or their representative parts are counted according to SCHRADER & GERSONDE (1978) and HORVAT (2004).Diatom measurements were made using free ImageJ software (https://imagej.nih.gov/ij/).
Determined diatoms are traditionally divided into two orders: Centrales (radial symmetry) and Pennales (bilateral symmetry).

Mineralogy
Xray powder diffraction (XRPD) was recorded on bulk samples, insoluble rock residue and the < 2 µm fraction of samples.Some samples contained large amounts of carbonate minerals that had to be dissolved to eliminate overlapping diffraction peaks with other minerals.Carbonate fractions were dissolved by acetic acid with an ammonium acetate (1 mol dm -3 ) buffer of pH 5 (JACK-SON, 1956).Clay minerals were determined on the < 2 µm fraction of samples on oriented mounts of air dried material, and after glycol treatment, heating to 400 ºC and 550 ºC (STARKEY et al., 1984).For quantitative analyses samples with 10% internal standard (zincite) were carefully weighed and ground with about 4 ml of methanol in a McCrone mill.
Quantitative analyses of the insoluble residue of samples were made using the RockJock ® computer program (EBERL, 2003).

Geochemistry
For better understanding of their mineralogical composition all selected samples were also analysed by XRF methods for the major oxides and some trace elements as Ba, Cu, Ni, Pb, Sr, V 2 O 5 , Zn, and Zr in laboratories of the Bureau Veritas Commodities Ltd (Vancouver, Canada).A 5 g of sample was heated to determine the loss on ignition (LOI).The treated sample was then fused in a platinum-gold crucible with a lithium tetraborate.The molten material is cast in a platinum mold.Fused discs were analysed on PANalytical Axios mAX .

Insecticidal efficacy
In order to test insecticidal efficacy of inert dusts against rice weevil, S. oryzae, 100 grams of clean soft wheat of different varieties (approx.13% m.c.) was mixed with a determined quantity of each dust in glass jars of 200 mL.Fifty unsexed adults of S. oryzae, 721 days old were added into each jar.Inert dusts were tested in 3 or 4 different doses, depending on inert dust, and all treatments were conducted in 4 repetitions.Bioassay was kept under controlled conditions at 28±2 °C, 65 ±5 % RH and in dark.
Insecticidal efficacy was estimated as lethal doses required to kill 50% and 90% of the tested population (LD 50 and LD 90 ).The LD 50 and LD 90 values were calculated by Probit analysis using IBM SPSS Statistics (IBM Corp. Released, 2013).
Diatom distribution with their sizes is given in Table 1. to emphasise the physical character of diatomite as a potential natural pesticide.The most abundant species in the samples belong to the Coscinodiscus group, Thalassionema nitzschoides and Chaetoceros group with resting spores and siliceous cysts.Based on palaeontological results, the most prospective diatomite (tripolite) for commercial use would be from Medvednica Mt. (MA4, PDK, D02B), Fig. 3.The dominant species in sample MA4 is  The measured content of trace elements of nine DE samples is given in Table 4.It is significant that the concentrations of Mo and Cu are higher in samples from Medvednica Mt. than form elsewhere.The highest concentration of Pb (19.4 mg/kg), which exceed the recommended value of 15 mg/kg for diatomaceous earth of standard sample, is determined in sample MA4 from Medvednica Mt. (Tab.4).The samples from Medvednica Mt. and samples MR-10 and MR-10B from Banovina region also have increased concentrations of Ni (>50 mg/kg).The elevated concentrations of uranium, especially in sample MR-10 from Banovina, are connected to the higher content of organic matter.

Insecticidal efficacy on common stored pests
Results among our samples of inert dust efficacy against rice weevil (RW), S. oryzae are shown in Table 6., where the highest efficacy is seen in samples D01, D02B and MA4.

DISCUSSION
The results of XRPD analyses showed that all analysed samples contain greater or lesser amounts of opal-A and quartz (Tab.2).Quantitative analyses of the mineralogical composition of samples obtained by XRPD analyses (Tab.2.) gave larger amounts of opal silica than results of calculations by Boströms' and Murdmaas' formulas (Tab.5).These types of calculations are based on the assumption that the SiO 2 /Al 2 O 3 ratio in terrigenous clay and silty clay is almost constant, and varies from 3.5 to 2.7.According to GRIZELJ et al. (2007;2017) XRPD analyses of Miocene pelitic sediments from the Croatian part of the PBS revealed that most of the samples contain the same mineral phases, but they are present in different quantities in different samples.The ratio of SiO 2 /Al 2 O 3 can vary within one clay mineral group and varies considerably within different clay mineral groups.Clay minerals have a wide range of variations in chemical composition and the variation is a composite of all the errors inherent in any Thalassionema nitzschioides in an assemblage with abundant smaller species of Coscinodiscus curvatulus, C. doljensis and bigger but mostly fragmented Thalassiosira leptopus.In the PDK dominant diatoms include Coscinodiscus oculus iridis, Thalassiosira leptopus, Coscinodiscus stellatis and Paralia sulcata.

Mineralogy
The mineralogical composition of the insoluble residues of the analysed rock samples is given in Table 2.The main components of all samples are carbonate minerals (calcite and/or aragonite), clay minerals and opaline silica (biogenic opal-A with very small amounts of mineral opal-C in some samples).Opal-A (highly disordered, near amorphous) produces a single broad peak centred at approximately 4 Å (Fig. 4) and opalC (wellordered αcri sto balite) at 4.044.06Å (JONES & SEGNIT, 1971).All analysed samples contain quartz (SiO 2 ) ranging in content from 15%.Some samples contain smaller amounts of feldspar and pyrite.analytic analysis: operator, instrument, method, beneficiation, sampling (WEAWER and POLLARD, 1973;WEAVER 1989).
According to the geochemical analyses there is a significant variation in the total silica (SiO 2 ) content in samples collected on  3).This is particularly evident at the Podsusedsko Dolje site, where the distance from the sample PDK and samples D01 and D02B is about a kilometre.Late rally marked variations in the silica content of the investigated diatomites are most likely the result of the marine geomorphology of the depth and its variations during the mid-Sarmatian, as well as diagenesis and tectonic processes that modify the composition and crystalline phase of the mineral.This also applies to the SiO 2 content at the Jurjevčani (18.65%) site (Žumberak Mt.), which is significantly lower than the average silica content on the Medvednica Mt. (mean = 48.63%)considering that the direct distance from the Podsusedsko Dolje site is only 25 km today.This is in accordance with their origin where the most prospective dia-  MA4 (Fig. 3,Tab. 1).The relatively high content of opalA (28%) and a small amount of quartz (5%) characterised sample MA4 (Tab.2).In previous testing (LIŠKA et al., 2015) of samples (MA4, MR10, MR10B, OP4, OP4A and PD 1) against Tribolium castaneum (HERBST) and Rhyzopertha dominica (FAB.) the highest efficacy (7 and 14 days after treatment) showed in sample MA4, reaching T. castaneum mortality of 61.50% and 99%, respectively, and 25.50% and 34.0%, respectively against R. dominica.In addition, there were no statistical differences of mortality rates with the standard Celatom ® MN 51.According to ZIAEE et al. (2013) each diatom species could have its own mode of physical action, regarding the number and size of pores and distribution of striae and if it is greater, the insecticidal activity would be higher.The results on diatom morphometrics show that the most efficient diatomites consist of mid sized (<45 μm) Coscinodiscus (Coscinodiscus curvatulus and C. doljensis) with numerous smaller pores, and lancet shaped Thalassionema nitzschioides that make up its active surface (Fig. 3).This is in agreement with KORUNIĆ (1998), that diatoms with discoid, flattened and linear body shapes better cover insect cuticle in relation to diatoms of cylindrical and round shapes.DE particles can effectively adsorb epicuticular lipids from the cuticle surface of the treated insects (ROHITHA PRASANTHA et al., 2015) and these phenomena may have a great effect on efficacy against insects.According to KORUNIĆ et al. (2016), larger available active surfaces have a higher sorption capacity for the lipids and are more effective desiccants.In the experiment conducted by KORUNIĆ & FIELDS (2016) silica aerogel Sipernat 50S with oil sorption of 3.23 ml/g dust was significantly more effective against Sitophilus granarius (LINNAEUS) in comparison with other DEs with oil sorption from 1.13 to 1.96 ml/g dust.Inert dusts MA4 with oil sorption of 1.23 and Celatom ® MN 51 with oil sorption of 1.20 were equally effective against rice weevil Sitophilus oryzae (LINNAEUS) and granary weevil, S. granarius.Nevertheless, noticeable sponge spicules and silicoflagellates in samples may damage the digestive tract or breathing organ, also resulting in internal desiccation or suffocation of the insects (JACKSON & WEBLEY, 1994), which increases efficacy as well.Sample MR-10 from Banovina region has only 3 % opal-A, while sample MR-10B did not contain opaline silica.Two samples from Slavonia OP4 and OP4A contain small amounts of opalC (Tab. 2).Based on the palaeontological results, the sporadic appearance of sponge spicules was noticed only in samples of OP, which is in agreement with the results of Murdmaas' modified formula (Tab.5).These samples are partially modified by calcinations in the process of diagenesis.It is well known that during diagenesis siliceous deposits undergo mineralogical changes from non-crystalline biogenic silica (opalA) to microcrystalline opal (opalCT/C) to microcrystalline quartz (SiO 2 ) (WILLIAMS et al., 1985;FLÖRKE et al., 1991;CADY et al., 1996;LYNNE & CAMP-BELL, 2004).Microcrystalline silica minerals (quartz) have been shown to be carcinogenic if inhaled.In addition, crystalline silica is classified as carcinogenic to humans by the International Agency for Research on Cancer (IARC, 1997).DEs registered as insecticides generally have less than 6% of quartz, like all analysed samples.However, the use of a proper dust mask, or the use of low crystalline silica DE can protect against this health risk (DESMARCHELIER & ALLEN, 2000).Very small amounts of opal-C were contained in samples PDK and D-01 (Tab.2).Sample PDK appeared to have the best outcome on RW with inert dust less than 25 microns even after 5 days.This is in agreement with the fact that smaller particles of DEs (<45 µm) are significantly more effective than larger particles (KORUNIĆ et al., 2011).Species are mostly smaller than usual, but this is in accordance with their origin in the upwelling zone and with seasonal changes during the Sarmatian, that limited their rapid growth in the blooming period (JURILJ, 1957;GALOVIĆ & BAJRAKTAREVIĆ, 2006).Nevertheless, sample PDK has a high content of carbonate minerals (calcite and aragonite), and a smaller amount of opal-A (12%), and quartz (3%) (Tab.2).Despite lower concentrations of amorphous silica in samples MA4 (28%) and PDK (12%), in combination with the increased content of smectite gives even better absorption properties.Clays are also known as inert dusts like DEs (MACELJSKI & KORUNIĆ, 1972;BANKS & FIELDS, 1995;SUBRAMANYAM & ROESLI, 2000) that also influenced better adsorptions here.In almost all the analysed samples smectite is the dominant clay mineral.It has a 2:1 layer structure, with a large base exchange capacity (60-130 meq/100 g) and will readily adsorb cations such as H + , Na + , Ca 2+ , and Mg 2+ (WEAVER, 1989).Interlayer cations are hydrated and replaceable, resulting in the swelling and dehydration characteristic for smectite (SLOVENEC & BERMANEC, 2003).

CONCLUSION
Sarmatian diatomites from Medvednica Mt. have different physical properties and efficacy on grain store insects.The analysed Croatian diatomites have significantly lower concentration of sili ca in relation to the standard DE.Diatomites from borehole (D01, D02) have the highest amorphous silica component (41 50%) and small amounts of quartz (45%) which makes them suitable for commercial usage as DE.They exhibit the highest efficacy on stored products insect rice weevil S. oryzae, together with inert dust from Medvednica Mt. (MA4, PDK).Based on palaeontolog ical results the most efficient analysed DE consists of midsized planktonic Coscinodiscus group of species where Thalassionema nitzschioides dominate which is positively correlated with their absorption.Even much smaller concentrations of amorphous sili ca in sample MA4 (28%) and PDK (12%), but in combination with increased content of smectite gives even slightly better absorption property.The higher level of CaO (9.6336.29) in correlation with Celatom ® MN 51 (5.60) probably also increased efficacy.Ca (II) ions are known to improve flow rate in modified diatomite (BAZHAL et al., 1975).Probably because of all mentioned para meters diatomite MA4 and smaller fraction of PDK show no diffe rences of mortality rates with the standard Celatom ® MN 51.Unfortunately, the small thicknesses of all prospective Croatian diato mites (approx.48 cm) and the slightly higher content of lead in MA4 sample make them inadequate for classical usage as DE (MA4= 19.4. mg/kg,Tab. 4).According to the standard sample Celatom ® MN 51 the allowed maximum Pb concentration is 15 mg/kg.The content of quartz in the samples is 15 %.Concerning insecticidal effects against the rice weevil, S. oryzae and taking into account the small thicknesses of all prospective Croatian diatomite layers, there is the possibility of mixing these inert dusts with some other natural compounds, with other insecticide effects, to provide the same (or even higher) insecticidal level.

ACKNOWLEDGEMENT
Financial support for this research was provided by the Croatian Science Foundation (HRZZ) through the scientific research project IP1120135570 (project leader Prof. dr.sc.Vlatka Rozman): Development of new natural insecticide formulations based on inert dusts and botanicals to replace synthetic, conventional insecticides, http://www.diacromixpest.eu/.The authors are grateful to Snježana MIHALIĆ ARBANAS, Faculty of Mining, Geology and Petroleum Engineering, for allowing using Dolje borehole samples for the purpose of this research.We would like to thank to Stjepan ĆORIĆ (GBA, Austria) and anonymous reviewer as well for their valuable comments and suggestions that improve the manuscript.

Figure 2 .
Figure 2. The parts of the geological outcrops from Medvednica Mt.where the most effective Croatian diatomite and tripolite were found.

Figure 3 .
Figure 3. SEM images of partially crushed diatom Thalassionema nitzschioides on the left an Coscinodiscus curvatulus on the right from the sample D-02B.

Table 1 .
continued and Zr.Additionally, volatization during fuming may result in some loss of As, S and Sb.The analyses were done by SPECTRO CIRIOS VISION ICPAES and Perkin Elmer Elan 6000/9000 ICP-MS.Inorganic carbon was determined by directly measuring the CO 2 gas evolved into the LECO CS230 analyzer when a prepared sample split is leached with perchloric acid.
®MN 51) the majority of analysed samples have a high content of CaO (> 35%) and relatively high content of MgO (Tab.3).The high content of carbonate component in the samples results in high values of LOI (>18.60%).

Table 3 .
Major and trace elements content measured by XRF method (in %) in Croatian diatomites and major elements in Celatom® Diatomaceous Earth Functional Additive Celatom ® MN 51 standard.
*LC50 and LD90 expressed as parts per million (ppm), Confidence limits (CL) are given in parentheses
(KORUNIĆ, 2013)AKTAREVIĆ, 2006)ong to the upwelling zone of the temperate climate with seasonal blooming(GALOVIĆ & BAJRAKTAREVIĆ, 2006), while that from Žumberak belongs to a more coastal marine area.The concentration of silica in the samples from Banovina is lower than that registered in the samples from the Medvednica and Žumberak Mts.(Tab.3).The lowest content of SiO 2 is found in the samples from Slavonia (OP: ≤7.37%) which is why we advocate tripolite instead of ordinary diatomite.The SiO 2 concentrations in those analysed samples are more depleted in comparison with the standard Celatom ® MN51.The concentration ranges from 3.96 to 7.37% SiO 2 (Tab.3).The insecticidal effect of DEs against stored product insects greatly depends on the amount of biogenic silica(KORUNIĆ, 2013).Samples with the lowest concentration of silica have the highest car-The Fe 2 O 3 content is slightly higher in all analysed samples from the Medvednica Mt. as well as in the sample MR-10B (Banovi na) in comparison to Celatom ® MN 51.The highest concentration of iron registered in the sample MA4 from the Medvednica Mt. probably originates from the chlorite mineral group occurring in the sample (Tab.2).The sum of all other oxides (Na 2 O, K 2 O + MnO + TiO 2 + P 2 O 5 ) in the analysed samples of Croatian diatomites is smaller than their content in Celatom ® MN 51 (Tab.3).Among the trace elements, only the concentrations of potentially toxic lead (Pb) and arsenic are of importance.The amount of Pb exceeds the limit values relative to Celatom ® MN 51 (15 mg/kg) solely in the sample MA4 (19.4 mg/kg) from Medvednica Mt., while the concentrations in all other samples are lower.Enhanced lead might be a result of the weathering of the galenite ore deposit there.The content of arsenic (As) is significantly lower in all analysed samples compared to sample Celatom ® MN 51 (Tab.4).The highest efficacy against the rice weevil, S. oryzae show ed in samples D01, D02B and MA4 (Tab.6).Samples D01 and D02B have smaller amounts of quartz (45 %) and maximum amount of opalA (4150%), as BSi, which make them suitable for commercial usage as DE (Tab.2,Fig.4).As a BSi component, Thalassionema nitzschioides dominate in the assemblage with abundant, bigger species (6580 μm) of Coscinodiscus group (Coscinodiscus oculus iridis, Thalassiosira leptopus, Coscinodiscus radiatus, Coscinodiscus stellatis, Coscinodiscus rothii, Coscinodiscus perforatus var.cellulosus), which are mostly fragmented, and with their smaller representatives (Coscinodiscus curvatulus, Coscinodiscus doljensis, Coscinodiscus sarmaticus, Coscinodiscus rugulosus, Paralia sulcata), giving better efficacy of D02B sample as DE like