2017 | 70/1 | 27–39 | 4 Figs. | 6 Tabs. | www.geologia-croatica.hr Journal of the Croatian Geological Survey and the Croatian Geological Society 1. INTRODUCTION Diatomite is a porous, lightweight, siliceous sedimentary rock mainly composed of diatom skeletons formed in marine to fresh- water environments of mid to high latitudes and contains >30% amorphous opaline silica (SiO2·nH2O). Commercial diatomite contains >60% of biogenic silica (BSi). Mostly high-grade diato- mite (>80% BSi) is used as diatomaceous earth (DE), crushed and oven-dried inert dust with a minor content of calcium car- bonate, 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 regis- tered in many countries as a food additive. DE is known as a po- tentially 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, Croatian diatomites and their possible application as a natural insecticide Ines Galović1, Josip Halamić1, Anita Grizelj1, Vlatka Rozman2, Anita Liška2, Zlatko Korunić3, Pavo Lucić2 and Renata Baličević2 1 Croatian Geological Survey, Department of Geology, Sachsova 2, 10000 Zagreb, Croatia; (ingalovic@hgi-cgs.hr) 2 University of Josip Juraj Strossmayer in Osijek, Faculty of Agriculture in Osijek, Vladimira Preloga 1, 31000 Osijek, Croatia 3 Diatom Research and Consulting Inc., 14 Tidefal Dr. Toronto, ON, MW 1J2, Canada doi:10.4154/gc.2017.04 Abstract In recent decades, there has been an increase in the use of diatomaceous earth (DE) as a natu- ral 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 mid- Miocene 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 diato- mites 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 Cro- atian 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 amor- phous 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 pa- laeontological 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 us- age 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 Keywords: diatomite, palaeontology, mineralogy, geochemistry, natural insecticide, mid-Miocene, Croatia chlorinated hydrocarbons, Bacilus thuringiensis, botanicals and chemicals (SUBRAMANYAM & HAGSTRUM, 1995; KLJAJIĆ & PERIĆ, 2005). Since only a physical method of controlling in- sects 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 re- placing 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 morpho- logical 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/cm3) 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 dehy- dration 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 Pod- susedsko Dolje outcrops (Medvednica Mt.), and can be described as diatomaceous tripolite (outcrops J1­J4: PANTOCSEK, 1903 G eo lo gi a C ro at ic a Geologia Croatica 70/128 System (PBS) (Central Paratethys) (PAVELIĆ, 2001; PAVE LIĆ et. al., 2003) (Fig. 1). This intracontinental region began to de- velop in the early Miocene as a result of the African and Euro- pean plate collision (HORVÁTH & ROYDEN, 1981). PBS is sur- rounded 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 Medi- terranean, Boreal and Indo­Pacific Oceans (STEININGER et al., 1988; RÖGL, 1996). These connections have been repeatedly dis- rupted and re-established until the end of the Badenian when the sea level dropped, causing the beginning of the isolation and dis- integration 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 fa- voured further decrease in salinity, resulting in changes to flora and fauna and their endemism. This was all more strongly influ- enced 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 Sarma- tian 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 frag- ments. 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 char- acteristic of the early post­rifting phase (PAVELIĆ et al., 2003). and JURILJ, 1957; outcrop B1: KOCHANSKY­DEVIDÉ & BAJ­ RAKTAREVIĆ, 1981; outcrops PD­MA: GALOVIĆ & BAJ­ RAK TAREVIĆ, 2006), and Lopatice and Gornja Šemnica in Hr- vatsko Zagorje (outcrops B2­B3: BAJRAKTAREVIĆ, 1984) (Fig. 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) and JURILJ (1957). During the investi- gations 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 Medved- nica, Žumberak (Jur­1 and Jur­2) and Požeška gora Mts. Seventy­ seven more diatom taxa were determined during those investiga- tions 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 Founda- tion (HRZZ) project „Development of new natural insecticide formulations based on inert dusts and botanicals to replace syn- thetic, conventional insecticides“ – DIACROMIXPEST con- ducted 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 re- sults of Croatian diatomites are presented here with their potential usage as DE in agriculture for the protection of stored products. 2. 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 Figure 1. Locations of investigated diatomites in Croatia, Pannonian Basin System based on http://maps-for-free.com:  – J1-J4 (1903 to 1957) and B1-B3 (1981 to 1984);  – Basic Geological Map of Republic of Croatia 1:50.000 from 2000 to 2009;  – this investigations with explanations in the text. G eologia C roatica I. Galović et al.: Croatian diatomites and their possible application as a natural insecticide 29 Figure 2. The parts of the geological outcrops from Medvednica Mt. where the most effective Croatian diatomite and tripolite were found. G eo lo gi a C ro at ic a Geologia Croatica 70/130 Since the beginning of the Miocene (23 Ma; OGG et al., 2016), the most commercially exploited diatomaceous earth de- posits are lacustrine in origin, although the largest producing de- posit 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 up- welling 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 environ- ment, thin-bedded to laminated marls and sandstones accumu- lated (VRSALJKO et al., 2005; VRSALJKO, 1997). In the NCB Sarmatian shallow-water gravels, biocalcare- nites, 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 Bade- nian deposits, (VRSALJKO, 2003). 3. MATERIALS AND METHODS 3.1. Field research with laboratory tests The representative sediments for diatom analyses belong to marl, tripolite, laminated marls, diatomite, varve, silt, clay and their varieties (Fig. 2). The thicknesses of the sampled diatomites are small, approx. 48 cm. They are mostly laminated as a result of seasonality. Among 26 well known Sarmatian outcrops of diato- mites in Croatia, twelve were selected for further investigation: Baničevac (BN) and Opatovac (OP) in Slavonia, Jurjevčani (JU) at Žumberak Mt., Crkovec (CR) in Hrvatsko Zagorje, Markuševec (MA) and Podsusedsko Dolje (PD) at Medvednica Mt. and Bačuga (BA), Martinovići (MR), Prnjavor Čuntički (PR), Šušnjar (SU), Mali Deanovići (MD) and Vukičevići (VU) in Banovina. Samples from the Dolje borehole at Podsusedsko Dolje were also examined (D) (ROZMAN et al., 2016) (Fig.1). In total, 57 samples (each of ca. 10 kg) were taken for preliminary testing on their insecticidal efficacy by comparing them with standard medium to high effec- tive Celatom® MN 51. After testing, ten samples showed promis- ing insecticidal efficacy and were selected for further analyses: MA­4, PD­1, PDK, D­01 and D­02B, JU­1, OP­4 and OP­4A (less laminated marls from the same horizont), MR10 (siltic marl) and MR-10B (mm laminated marl) and examined as natural pesticide (LIŠKA et al., 2015). Sample PDK is mixture of all five PD sam- ples, but milled to a diameter <45 μm for testing efficacy with smaller particles in the second year of the research project. Based on their preliminary physico-chemical properties (palaeonto lo- gical, mineral and geochemical analyses) only samples from Med- vednica Mt. (Fig. 2) were affirmed as diatomite. 3.2. Palaeontology The standard preparation method used by the Croatian Geologi- cal Survey is applied. Approximately 1 cm3 of the sediment is placed in a beaker and treated with 30% concentrated hydrogen peroxide (H2O2) 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 re- move 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 magnifica- tion (VILIČIĆ, 2003; HORVAT, 2004). Only whole diatom valves or their representative parts are counted according to SCHRADER & GERSONDE (1978) and HORVAT (2004). Diatom measure- ments were made using free ImageJ software (https://imagej.nih. gov/ij/). Determined diatoms are traditionally divided into two or- ders: Centrales (radial symmetry) and Pennales (bilateral sym- metry). 3.3. 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 frac- tion of samples on oriented mounts of air dried material, and af- ter 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. For XRPD analysis a Philips vertical X­ray goniometer (type X‘Pert) equipped with a Cu tube at the Croatian Geological Sur- vey (Zagreb, Croatia) was used. Experimental conditions were: 45 kV, 40 mA, PW 3018/00 PIXcel detector, primary beam diver- gence 1/4 º, and continuous scan (0.02 º2Θ/s). Quantitative analyses of the insoluble residue of samples were made using the RockJock® computer program (EBERL, 2003). 3.4. Geochemistry For better understanding of their mineralogical composition all selected samples were also analysed by XRF methods for the ma- jor oxides and some trace elements as Ba, Cu, Ni, Pb, Sr, V2O5, 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 AxiosmAX. The inductively coupled plasma atomic emission spectrom- etry/inductively coupled plasma emission mass spectrometry (ICP­AES / ICP­MS) methods for measuring trace elements were performed on 15 g samples. The 0.25 g split were heated in HNO3-HClO4­HF to fuming and taken to dryness. The residue was dissolved in HCl. This preparation method is only partial for some Cr and Ba minerals and oxides of Al, Fe, Hf, Mn, Sn, Ta G eologia C roatica I. Galović et al.: Croatian diatomites and their possible application as a natural insecticide 31 Table 1. Diatom distribution with their morphometrics. Legend: + scattered (< 5 %), rare (5–9 %), low abundance (10–15%), abundant (16–35%), high abundance (36–50%), dominant species (>50%) – species in fragments. Diatom/sample PD-1 D-01 D-02B MA-4 JU-1 PDK size (μm) Centrales               Actinocyclus octanarius EHRENBERG   + +     + 8–35 Actinocyclus octanarius var. tenellus (BREB.) HENDEY   +     + + 25–60 Actinoptychus senarius (EHRENBERG) EHRENBERG   + +     + 20–62 Anaulus minutus GRUNOW +         + 10–16 Anaulus simplex HAJÓS +   +   + + 8–12 Asteromphalus brunii PANTOCSEK   +         32–44 Coscinodiscus group (Coscinodiscus, Hyalodiscus, Melosira, Paralia, Stephanopyxis, Thalassiosira) + + 22 25 16 27   Coscinodiscus apiculatus EHRENBERG     +       – Coscinodiscus curvatulus GRUNOW + + 8 +   + 19– 37 Coscinodiscus doljensis PANTOCSEK   + + 17 + + 28–43 Coscinodiscus miocaenicus KRASSKE   +   +     18–21 Coscinodiscus obscurus SCHMIDT     +       38 Coscinodiscus oculus iridis EHRENBERG +   + + + + ≥40–75 Coscinodiscus perforatus var. cellulosus GRUNOW     +     + – Coscinodiscus radiatus EHRENBERG   + +       68–72 Coscinodiscus rothii (EHRENBERG) GRUNOW   + + +     39–80 Coscinodiscus rugulosus HAJÓS   + +       14–30 Coscinodiscus sarmaticus PANTOCSEK     + + + + 12–31 Hemiaulus cf. polymorphus           + 46 Hyalodiscus laevis EHRENBERG           + 20–50 Hyalodiscus scoticus (KÜTZING) GRUNOW         + + 10–40 Melosira dickiei var. fossilis PANTOCSEK     +       12–20 Melosira sp.         +   – Paralia sulcata (EHRENBERG) CLEVE + + + + + 7 12–40 Perissonoë trigona (GRUNOW) ANDREWS & STOELZEL           + – Pseudopodosira westii (SMITH) SHESHUKOVA-PORETZSKAYA & GLEZER     + +   + 12–29 Rhizosolenia oligocaenica SCHRADER   + + + + + – Stellarima stellaris (ROPER) HASLE & SIMS + + + + +   36 Stephanopyxis turris (GREVILLE) RALFS +   +       63 Thalassiosira leptopus (GRUNOW) HASLE & FRYXELL + + + + + + 34–65 resting spores and siliceous cysts (Bacteriastrum spp., Chaetoceros spp, Liradiscus spp., Xantiopyxis spp., Periptera spp.) 6 59 + + 6 6 – Triceratium balearicum CLEVE & GRUNOW           + – T. laetum fa. quadrata HAJÓS           + 28 Pennales               Achnanthes baldjiki (BRIGHTW.) GRUNOW           + 32 Achnanthes brevipes AGARDH   +         31 Achnanthes rara JURILJ   +       + 29–34 Achnanthes saeptata var. sussedana JURILJ         +   50 Achnanthes spp.         +   – Amphora binodis var. biggiba GRUNOW +         + 28 Amphora costata SMITH +         + 63 Ardisonia fulgens (GREVILLE) GRUNOW           + – Biddulphia biddulphiana (BOYER) SMITH     +   +   54–117 Biddulphia sp.           + – Caloneis boryana PANTOCSEK           + 42 Climacosphenia moniligera EHRENBERG           + 16–18 Cocconeis andesitica PANTOCSEK           + 30 C. canaliculata JURILJ           + 15 Cocconeis conciata PANTOCSEK +         + 26 C. disculus (SCHUMANN) CLEVE           + 14 C. distans GREGORY           + 40 C. evolvens JURILJ           + 23 Cocconeis fluminensis (GRUNOW) PERAGALLO +         + 28 C. quarnerensis var. lanceolata JURILJ           + 20 Cocconeis cf. sarmatica PANTOCSEK +         + 11–28 Cocconeis scutellum EHRENBERG + + + + + 5 9–45 Cymatosira lorenziana var. maior JURILJ +         + 34–54 Delphineis angustata (PANTOCSEK) ANDREWS   +         20–35 Delphineis surirella (EHRENBERG) ANDREWS   + +       20 Dimeregramma angustatum HAJÓS         5   40 G eo lo gi a C ro at ic a Geologia Croatica 70/132 Diatom/sample PD-1 D-01 D-02B MA-4 JU-1 PDK size (μm) Centrales               Dimerogramma boryanum PANTOCSEK           + – Dimeregramma distans GREGORY         +   39 Di. minor (GREGORY) RALFS           + 12–17 Di. minus var. neglectum JURILJ           + – Diploneis bombus EHRENBERG         +   – Diploneis coffaeiformis (SCHMIDT) CLEVE           + – Diploneis crabro (EHRENBERG) EHRENBERG     + +   + 22–45 Diploneis fusca (GREGORY) CLEVE +         + 51 Diploneis ovalis (BRÉBISSON) CLEVE + + + +   + 34 Diploneis splendida var. porosa JURILJ       +     – Diploneis subovalis CLEVE     +     + 43–57 Diploneis smithii (BRÉBISSON) CLEVE +       + + 30–50 Diploneis sp.   +         – Encyonema sp.           + – Epithemia adnata (KÜTZING) BRÉBISSON         +   46 Epithemia zebra var. saxonica (KÜTZING) GRUNOW           + – Eunotia cf. tenella (GRUNOW) HUSTEDT           + – Fragilaria brevistriata GRUNOW +     +   + 9–15 Fragilaria construens (EHRENBERG) GRUNOW     +     + 8 Grammatophora angulosa EHRENBERG       +   + 28–32 Grammatophora insignis GRUNOW +       + + – Grammatophora macilenta SMITH         +   25–80 Grammatophora miocaenica HAJÓS   +         28 Grammatophora robusta EHRENBERG       +     40–60 Grammatophora oceanica EHRENBERG   + +     + 37–93 G. oceanica var. macilenta (SM.) GRUNOW           + do 172 G. oceanica var. oceanica EHRENBERG           + – Grammatophora stricta EHRENBERG + +     + + 22–40 G. stricta var. biharensis EHRENBERG           + – Hantzschia virgata (ROPER) GRUNOW         +   50–66 Lyrella hennedyi (W.SMITH)STICKLE & MANN + +       + 25–45 Mastogloia binotata (GRUNOW) CLEVE         +   40 Mastogloia lacustris (GRUNOW) GRUNOW     +   +   38 Mastogloia sarmatica JURILJ +   +     + 69 M. splendida (GREGORY) CLEVE           + – Mastogloia sp. + + +   9   – Navicula latissima var. quadrata JURILJ       +   + 64 Navicula spp.   +     +   – Nitzschia doljensis PANTOCSEK   +         35 Nitzschia frustulum (KÜTZING) GRUNOW     + +     18 Nitzschia spp. +       +   – Opephora gemmata fa. minor JURILJ         +   40 Opephora marina (GREGORY) PETIT           + 17 Plagiogramma biharense PANTOCSEK     +       15 Plagiogramma bipunctatum HAJÓS +           – Plagiogramma staurophorum (GREGORY) HEIBERG +   +   + + 20–30 Planothidium quarnerensis (GRUNOW) WITKOWSKI,LANGE-BERTALOT & METZELIN         + + 20 Rhabdonema hamuliferum KITTON         12   – Rhaphoneis amphiceros EHRENBERG         + + 17–32 Rh. amphiceros var. rhombica GRUNOW           + – Rh. cocconeiformis (SCHMIDT) HANNA & GRANT           + – Rh. nitida (GREGORY) GRUNOW           + – Rhaphoneis rhombica (GRUNOW) ANDREWS +         + 34 Rhopalodia gibberula (EHRENBERG) MÜLLER +       + + 34 Staurosirella leptostauron (EHRENBERG) WILLIAMS & ROUND         +   10–26 Staurosirella pinnata (EHRENBERG) WILLIAMS & ROUND   +         22 Surirella subfastuosa PANTOCSEK         +   – Synedra crystallina var. fossilis PANTOCSEK           + – Synedra fulgens (GREVILLE) W.SMITH +   +   +   – Synedra tabulata var. obtusa PANTOCSEK         +   – Thalassionema nitzschioides (GRUNOW) GRUNOW 69 32 74 60 30 8 33–139 Thalassiotrix longissima CLEVE & GRUNOW 8   + + + + 34 Table 1. continued G eologia C roatica I. Galović et al.: Croatian diatomites and their possible application as a natural insecticide 33 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 measur- ing the CO2 gas evolved into the LECO CS230 analyzer when a prepared sample split is leached with perchloric acid. From the last century, the Boströms’ standard formula (BOS- TRÖM at al., 1972) for estimating biogenic silica from geochemi­ cal data was used: Opal-A= SiO2 (total) – 3·Al2O3 We tested and abandoned this formula because of a negative result for sample MR­10B, (Tab. 5). Therefore Murdmaas’ modi­ fied formula (MURDMAA et al., 1980) for extracting opal­A from hemipelagic sediments was used: Opal-A= SiO2 (total) – 2.7∙Al2O3 3.5. Insecticidal efficacy In order to test insecticidal efficacy of inert dusts against rice weevil, S. oryzae, 100 grams of clean soft wheat of different va- rieties (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 (LD50 and LD90). The LD50 and LD90 values were calculated by Probit analysis using IBM SPSS Statistics (IBM Corp. Released, 2013). 4. RESULTS 4.1. Palaeontology In total, in samples from Medvednica and Žumberak Mts, 111 diatom species were determined with fourteen variants and two forms. Among them, four species had not been previously dis- covered in Podsusedsko Dolje: Chaetoceros didymus EHREN- BERG, Dimerogramma minus (GREGORY) RALFS var. neglec- tum JURILJ, Rhaphoneis cocconeiformis (SCHMIDT) HANNA & GRANT and Rh. nitida (GREGORY) GRUNOW. Approxi- mately 90% of the diatom species are less than 50 μm in size, while larger specimens are mostly broken (GALOVIĆ et al., 2015). Diatom distribution with their sizes is given in Table 1. to emphasise the physical character of diatomite as a potential natu- ral 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 (tripo- lite) for commercial use would be from Medvednica Mt. (MA­4, PDK, D­02B), Fig. 3. The dominant species in sample MA­4 is 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 2. Quantitative mineral composition of samples (in wt. %) obtained by XRD analysis using RockJock® computer program (EBERL, 2003). ** – abundant (20–40%), * – subordinate (1–20%), + – traces (<1%) MINERALS MA-4 PDK D-01 D-02B JU-1 OP-4 OP-4A MR-10 MR-10B Quartz 5 3 5 4 2 2 1 2 2 Opal-A 28 12 41 50 11 3 Opal-C + + 1 1 Calcite 19 30 10 11 28 7 6 69 64 Aragonite 34 40 73 81 Feldspar 1 2 1 1 1 1 Pyrite 2 Clay minerals 47 19 44 33 18 16 10 25 34 Smectite ** * ** ** * * * * * Illit/muscovite * * * * * * * * * Chlorite * + Kaolinite * + + + + G eo lo gi a C ro at ic a Geologia Croatica 70/134 4.3. Geochemistry The results of geochemical analysis for major elements are shown in Table 3. The concentration of total silica in samples varies from 3.96 to 61.91%. The measured content of SiO2 in most samples is relatively small (< 47.71%) except for the samples from the bore- hole Podsusedsko Dolje (D­01 and D­02B; 55.58 % and 61.91% respectively). In comparison to the standard sample (Celatom® 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%). 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 in- creased concentrations of Ni (>50 mg/kg). The elevated concen- trations of uranium, especially in sample MR-10 from Banovina, are connected to the higher content of organic matter. 4.4. Insecticidal efficacy on common stored pests Results among our samples of inert dust efficacy against rice wee- vil (RW), S. oryzae are shown in Table 6., where the highest ef- ficacy is seen in samples D­01, D­02B and MA­4. 5. 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 sam- ples obtained by XRPD analyses (Tab. 2.) gave larger amounts of opal silica than results of calculations by Boströms’ and Murd- maas’ formulas (Tab. 5). These types of calculations are based on the assumption that the SiO2/Al2O3 ratio in terrigenous clay and silty clay is almost constant, and varies from 3.5 to 2.7. Ac- cording to GRIZELJ et al. (2007; 2017) XRPD analyses of Mio- cene 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 SiO2/Al2O3 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, Thalas- siosira leptopus, Coscinodiscus stellatis and Paralia sulcata. 4.2. 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 dis- ordered, near amorphous) produces a single broad peak centred at approximately 4 Å (Fig. 4) and opal­C (well­ordered α­cri sto­ balite) at 4.04­4.06 Å (JONES & SEGNIT, 1971). All analysed samples contain quartz (SiO2) ranging in content from 1­5%. Some samples contain smaller amounts of feldspar and pyrite. 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. Sample/ Element SiO2 Al2O3 Fe2O3 CaO MgO Na2O K2O MnO TiO2 P2O5 LOI SUM*** of trace elements SUM TOT/C MA-4 47.71* 8.81 3.85 9.63 0.92 0.37 1.43 0.07 0.41 0.10 23.44 2.218 98.96 9.65 PDK 21.26 4.51 1.98 36.29 0.67 0.47 0.59 0.03 0.25 0.11 33.33 0.002 99.51 – D-01 55.58 6.83 2.85 5.59 1.30 0.25 1.05 0.04 0.31 0.21 22.52 2.442 98.97 8.56 D-02B 61.91 5.31 2.20 6.13 1.13 0.19 0.82 0.02 0.27 0.06 18.60 2.326 98.97 5.83 JU-1 18.65 3.15 1.34 38.10 0.49 0.21 0.44 0.04 0.16 0.10 35.22 1.048 98.95 10.07 OP-4 7.37 2.32 0.97 44.72 0.34 0.48 0.19 0.005 0.12 0.007 41.31 1.345 99.24 10.76 OP-4A 3.96 1.31 0.40 49.26 0.24 0.39 0.09 0.02 0.07 0.11 42.78 1.105 99.74 11.61 MR-10 14.21 4.73 1.57 37.15 1.89 0.24 0.83 0.07 0.24 0.08 36.53 1.541 99.08 10.42 MR-10B 16.22 5.44 2.05 36.90 1.42 0.16 0.92 0.07 0.23 0.07 35.04 0.728 99.25 9.93 Celatom® MN 51** 73.60 7.80 1.80 5.60 0.30 SUM other oxides 2.30 – 5.50 – 96.90 – * In % ** Standard DE *** Measured trace elements: Ba, Cu, Ni, Pb, SO3, Sr, V2O5, Zn, and Zr. Table 4. Trace element content in Croatian diatomites (mg/kg). Sample/ Element MA-4 PDK D-01 D-02B JU-1 OP-4 OP-4A MR-10 MR-10B Mo 24.4 17.4 20.2 14.4 2.2 2.3 0.8 98.7 8.0 Cu 46.8 32.9 41.5 82.9 21.3 24.6 31.0 37.9 31.4 Pb 19.4 6.9 14.7 11.8 5.6 10.8 4.3 10.4 12.9 Zn 75 33 65 63 27 23 14 48 52 Ni 113 81 89 81 31 35 13 133 62 Co 21 6 16 8 5 5 2 11 5 As 15 7 7 5 10 5 0.05 5 6 U 8 10 6 4 5 8 6 36 8 Th 8 3 6 4 2 2 1 3 3 Sr 518 3405 427 549 3438 6405 5161 1745 1653 Cd 0.8 6.2 0.8 4.5 0.8 1.1 0.3 2.9 1.6 Sb 1.8 0.9 1.3 1.7 0.6 2.2 0.4 4.1 2.0 Bi 0.5 <0.1 0.3 0.2 0.1 0.2 0.1 0.2 0.2 V 104 64 75 81 43 23 17 51 60 La 24 12 17 12 8 9 5 10 12 Ba 275 340 183 168 336 176 170 632 679 W 1.4 <0.5 1.2 0.8 0.5 0.4 0.2 0.6 0.6 Ce 54 21 35 23 15 17 9 20 24 Sn 2.4 1.0 1.6 1.3 0.7 0.8 0.6 1.1 1.3 Y 19 13 14 9 6 10 7 7 8 Nb 6 4 5 4 2 2 1 3 4 Ta 0.5 0.3 0.4 0.3 0.2 0.1 0.05 0.2 0.2 Sc 12 5 8 7 3 3 2 6 7 Li 40 23 35 29 14 12 9 25 30 Se 5 3 4 3 2 3 3 11 4 Zr 39 49 34 26 22 8 6 22 25 Celatom® Diatomaceous Earth Functional Additive Standard Celatom® MN 51: Pb = 15 mg/kg, As = 20 mg/kg. G eologia C roatica I. Galović et al.: Croatian diatomites and their possible application as a natural insecticide 35 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 (SiO2) content in samples collected on Figure 4. XRPD pattern of bulk sample D-02B, and insoluble rock residue of sample MA-4. Table 6. Efficacy of 9 Croatian inert dusts and standard Celatom® MN 51 against Sitophilus oryzae (L.) adults after 7 days of exposure with LD50/LD90 values (95% fiducial limits). Inert dust Lethal doses (ppm) LD50* LD90* D-01 156.5 (62.28-213.26) 447.2 (419.88-481.90) D-02B 300 ppm=82.5% 359.6 (240.55-422.56) JU-1 351.4 (324.22-373.43) 606.7 (575.90-648.08) MA-4 232.2 (183.05-266.05) 458.7 (436.21-487.52) MR-10 693.3 (646.91-778.77) 954.1 (847.50-1159.46) MR-10B 588.6 (564.40-623.79) 831.1 (763.36-944.09) OP-4 421.5 (346.71-460.50) 867.7 (750.76-1161.96) OP-4A 534.8 (517.44-555.17) 744.4 (699.14-813.40) PD-1 362.8 (314.80-391.50) 726.4 (668.39-816.94) Celatom® MN 51 300 ppm=83.5% 334.1 (86.64-399.00) *LC50 and LD90 expressed as parts per million (ppm), Confidence limits (CL) are given in parentheses Table 5. Content of Opal-AB according to standard Boströms’ formula (BOSTRÖM at al., 1972) and Opal-AM according to modified Murdmaas’ formula (MURDMAA et al., 1980). SiO2 and Al2O3 are from Table 3. Element/ mineral M A- 4 PD K D -0 1 D -0 2B JU -1 O P- 4 O P- 4A M R- 10 M R- 10 B SiO2 47.71 21.26 55.58 61.91 18.65 7.37 3.96 14.21 16.22 Al2O3 8.81 4.51 6.83 5.31 3.15 2.32 1.31 4.73 5.44 3∙Al2O3 26.43 13.53 20.49 15.93 9.45 6.96 3.93 14.19 16.32 2.5∙Al2O3 23.79 12.18 18.44 14.34 8.50 6.26 3.54 12.77 14.69 Opal-AB 21.28 7.73 35.09 45.98 9.20 0.41 0.03 0.02 –0.10 Opal-AM 23.92 9.08 37.14 47.57 10.15 1.11 0.42 1.44 1.53 G eo lo gi a C ro at ic a Geologia Croatica 70/136 the Medvednica Mt. (21.26% sample PDK, 47.71%, sample MA­ 4, and 61.91% sample D­02B) (Tab. 3). This is particularly evident at the Podsusedsko Dolje site, where the distance from the sam- ple 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 geomorpho- logy of the depth and its variations during the mid-Sarmatian, as well as diagenesis and tectonic processes that modify the com- position and crystalline phase of the mineral. This also applies to the SiO2 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 dis- tance from the Podsusedsko Dolje site is only 25 km today. This is in accordance with their origin where the most prospective dia- tomites from Medvednica Mt. belong 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 sam- ples from the Medvednica and Žumberak Mts. (Tab. 3). The low- est content of SiO2 is found in the samples from Slavonia (OP: ≤7.37%) which is why we advocate tripolite instead of ordinary diatomite. The SiO2 concentrations in those analysed samples are more depleted in comparison with the standard Celatom® MN­51. The concentration ranges from 3.96 to 7.37% SiO2 (Tab. 3). The insecticidal effect of DEs against stored product insects greatly depends on the amount of biogenic silica (KORUNIĆ, 2013). Sam- ples with the lowest concentration of silica have the highest car- bonate component (CaO) content. According to FIELDS & MUIR (1995) calcium oxide also has some insecticidal activity. Samples JU, OP and MR have enhanced CaO (36.90­49.26%) which could be a reason why they showed promising insecticidal efficacy in preliminary testing. All samples except D-01 and D-02B have much higher contents of CaO compared to the standard sample (Tab. 3). The MgO content is in most samples associated with the CaO content which is probably due to the originally precipitated dolo- mite in association with calcite. The content of alumina depends on the amount of the clay component in the sample. In relation to Celatom® MN 51 only one analysed sample, MA­4, has a slightly elevated content of alumina (Al2O3), which is probably a part of the detected smectite which increased its adsorption (Tab. 2 and 3). The Fe2O3 content is slightly higher in all analysed samples from the Medvednica Mt. as well as in the sample MR-10B (Bano- vi na) in comparison to Celatom® MN 51. The highest concentra- tion 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 (Na2O, K2O + MnO + TiO2 + P2O5) in the analysed samples of Croatian diato- mites 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 ana- lysed 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 (Co- scinodiscus oculus iridis, Thalassiosira leptopus, Coscinodiscus radiatus, Coscinodiscus stellatis, Coscinodiscus rothii, Coscino- discus perforatus var. cellulosus), which are mostly fragmented, and with their smaller representatives (Coscinodiscus curvatulus, Coscinodiscus doljensis, Coscinodiscus sarmaticus, Coscinodis- cus rugulosus, Paralia sulcata), giving better efficacy of D­02B sample as DE like MA­4 (Fig. 3, Tab. 1). The relatively high con- tent of opal­A (28%) and a small amount of quartz (5%) charac- terised 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 treat- ment) showed in sample MA­4, reaching T. castaneum mortality of 61.50% and 99%, respectively, and 25.50% and 34.0%, respec- tively 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 insecti- cidal activity would be higher. The results on diatom morpho- metrics 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 Tha- lassionema 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 cu- ticle in relation to diatoms of cylindrical and round shapes. DE particles can effectively adsorb epicuticular lipids from the cuti- cle surface of the treated insects (ROHITHA PRASANTHA et al., 2015) and these phenomena may have a great effect on effi- cacy 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 con- ducted by KORUNIĆ & FIELDS (2016) silica aerogel Sipernat 50S with oil sorption of 3.23 ml/g dust was significantly more ef- fective against Sitophilus granarius (LINNAEUS) in comparison with other DEs with oil sorption from 1.13 to 1.96 ml/g dust. In- ert 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 sil- iceous deposits undergo mineralogical changes from non-crystal- line biogenic silica (opal­A) to microcrystalline opal (opal­CT/C) to microcrystalline quartz (SiO2) (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 G eologia C roatica I. Galović et al.: Croatian diatomites and their possible application as a natural insecticide 37 insecticides generally have less than 6% of quartz, like all ana- lysed 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). Sam- ple 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 signifi- cantly more effective than larger particles (KORUNIĆ et al., 2011). Species are mostly smaller than usual, but this is in accord- ance 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). De- spite 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+, Ca2+, and Mg2+ (WEAVER, 1989). Interlayer cations are hydrated and replaceable, resulting in the swelling and dehydration characte- ristic for smectite (SLOVENEC & BERMANEC, 2003). 6. CONCLUSION Sarmatian diatomites from Medvednica Mt. have different physi- cal 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 suit- able 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 absorp- tion 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. Unfor- tunately, 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 lay- ers, there is the possibility of mixing these inert dusts with some other natural compounds, with other insecticide effects, to pro- vide 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 proj- ect IP­11­2013­5570 (project leader Prof. dr. sc. Vlatka Rozman): Development of new natural insecticide formulations based on inert dusts and botanicals to replace synthetic, conventional in- secticides, http://www.diacromixpest.eu/. The authors are grate- ful to Snježana MIHALIĆ ARBANAS, Faculty of Mining, Ge- ology 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 re- viewer as well for their valuable comments and suggestions that improve the manuscript. REFERENCES BAJRAKTAREVIĆ, Z. (1984): The application of microforaminiferal association and nannofossils for biostratigraphic classification of the Middle Miocene of N. Croatia.– Acta Geologica, 49/1, 1–34. BAJRAKTAREVIĆ, Z., BLAŠKOVIĆ, I. & POLŠAK, A. 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