Limited information and data are available on the material and structural performance of GC incorporating lightweight fine aggregate. In this research, three types of lightweight fine materials were utilized to partially replace sand volume of GC. These lightweight materials were rubber, vermiculite, or lightweight expanded clay aggregate (LECA) and they were used in contents of 20%, 40%, 60%, and 100%. The variables were applied to better investigate the efficiency of each lightweight material in GC and to recommend GC mixes for structural applications. The concrete workability, compressive strength, indirect tensile strength, freezing and thawing performance, and impact resistance were measured in this study. In addition, three reinforced concrete slabs were made from selected mixes with similar compressive strength of 32 MPa and then tested under a 4-point bending loading regime. The results showed that using LECA as sand replacement in GC increased its compressive strength at all ages and all replacement ratios. Compared with the control GC mix, using 60% LECA increased the compressive strength by up to 44%, 39%, and 27%, respectively at 3, 7, and 28 days. The slabs test showed that partial or full replacement of GC sand adversely affected the shear resistance of concrete and caused premature failure of slabs. The slab strength and deflection capacities decreased by 9% and 30%, respectively when using rubber, and by 23% and 59%, respectively when using LECA, compared with control GC slab. The results indicated the applicability of GC mix with 60% LECA in structures subjected to axial loads. However, rubber would be the best lightweight material to recommend for resisting impact and flexural loads. The use of Portland cement concrete has been questioned in recent decades. This is due to the high rate of fossil fuel and limestone  consumption, in addition to the high emission of carbon dioxide when Portland cement is produced ,2,3. TheThe development of high-rise concrete buildings and the associated infrastructure, increase the concrete demand day after day, which raises concerns about the future sustainable use of Portland cement concrete . Geopoly3, but with limited compressive strength of about 16 MPa. The highest compressive strength was found when using fine lightweight aggregate of 2.3 times that of the coarse lightweight aggregate. Bhogayata et al. [Lightweight concrete is an economical type of concrete that can benefit structures through higher strength to weight ratio, lower cost of foundation, and improved resistance to fire ,14,15,16a et al.  replacedNano materials have also been shown to enhance the GC performance . Raza etFew studies have been carried out on the structural application of GC in reinforced slabs. Meng et al.  tested GAs per the above literature, limited information and data are available on the effect of using lightweight fine aggregate as a partial replacement of GC sand on its performance at the material and structural levels. To address this research gap and to develop more sustainable concrete, eleven GC mixes were produced, tested, compared, and analyzed. The concrete mix properties, durability, and the structural behavior were investigated. The structural performances of reinforced GC slabs made of selected mixes and subjected to flexural load were also examined. The research aims to better investigate the efficiency of the lightweight aggregate in GC and to recommend GC mixes for structural applications.2 + Al2O3 + Fe2O3 is more than 70% of fly ash and hence it is classified as Class F. The specific gravities of fly ash and slag were 2.57 and 2.83, respectively. The coarse and fine aggregates used in all mixes were 10 mm dolomite stone and river sand with maximum aggregate size of 4.75 mm, respectively. Three lightweight fine aggregates  were used as partial replacement of GC sand. The aggregate particle sizes ranged between 2.36 mm and 4.75 mm for rubber, 0.3 mm and 2.36 mm for vermiculite, and 0.3 mm and 4.75 mm for LECA. 3, 2.71, and 7.89, respectively for dolomite; 1420 kg/m3, 2.61, and 2.20, respectively for sand; 530 kg/m3, 0.97, and 4.85, respectively for rubber; 80 kg/m3, 0.24, and 2.97, respectively for vermiculite; and 880 kg/m3, 1.7, and 4.3, respectively for LECA. High range water reducer (superplasticizer—SP) and retarder with respective specific gravities of 1.085 and 1.057 were used in this study. A mix of sodium silicate (SS) and sodium hydroxide (SH) solutions was used as the activator in the GC mixes. The specific gravity of the alkaline solution was 1.28 and the mixing ratio of SS:SH was 1.7:1.0 by weight. The prepared 12 M sodium hydroxide solution was left to cool down for 2 h and then mixed with sodium silicate solution for 30 min using a mechanical liquid stirrer. The activator was left to cool down to room temperature for 24 h before use in the concrete mixing.The binder materials used in all mixes were ground granulated blast furnace slag (GGBFS) and fly ash with chemical composition shown in 3, constant activator content of 207 kg/m3, constant water content of 177 kg/m3, constant fly ash content of 206 kg/m3, constant slag content of 206 kg/m3, constant superplasticizer content of 9.4 kg/m3, and constant retarder content of 2.1 kg/m3. The sand content in the GC control mix was 611 kg/m3 and was partially replaced with rubber, vermiculite, or LECA in different mixes by volume. The fine to coarse aggregate ratio was 1:1.GC mixes were designed with constant dolomite content of 611 kg/mThe mixing procedure was as follows: mix dry sand and stone for 1 min.; add all water and mix for 1 min.; rest for 2 min.; add fly ash and slag and mix while adding the activator, superplasticizer, and retarder in 3 min and mix for additional 2 min. when all materials are in; rest for 5 min.; and mix for the final 2 min. It was observed that the GC appeared flowable after adding all materials and mixing for additional 2 min.; however, that flowability quickly decreased within 5 min. This was the reason for leaving the mixes for 5 min. to rest and then mixing them again before measuring the fresh concrete properties and filling the prepared moulds. The variables in this investigation were: lightweight fine material , and sand replacement ratio . The ingredients of concrete mixes in this study are shown in Mechanical and durability characteristics were measured in this study including workability, unit weight, compressive strength , indirect tensile strength, freezing-thawing influence, and impact resistance. From each mix, two 100 × 200 mm cylinders per measurement day were prepared to measure both the unit weight and the 3, 7, and 28 day compressive strength. Another two 100 × 200 mm cylinders were prepared to measure the 28 day indirect tensile strength. A standard hammer and rod were used in compacting the prepared concrete specimens. The concrete was left in the moulds for 24 hrs before de-moulding, labelling, and curing were performed according to AS1012.8.1 .3 concrete cubes. The cubes were cut at 28 days concrete age from three 100 × 100 × 470 mm concrete prisms that were taken from each mix and kept in wet curing all the time until the test day. Half of the prepared cubes per mix were used for the freezing-thawing cycles and the other half were kept as control specimens. Three sets of freezing-thawing cycles  were applied with a 4 hrs period each full cycle. For each freezing-thawing cycle, the specimens were picked up from the curing water and their surfaces were dried using a piece of cloth, then they were inserted in a freezer with a temperature of −18 °C for 2 h (freezing time). The specimens were then fully surrounded by water in a container and moved to a fridge with a temperature of 4 °C for 2 hrs (thawing time). At the end of each cycle set, the compressive strength was measured using two cubes and compared with another two control cubes that were left in clean tap water from after the 28 days until the end of each cycle set (test day). The freezing-thawing effect and the impact resistance were measured for mixes GC, 60 R, 60 V, and 60 L. The freezing-thawing effect on GC compressive strength was measured according to ASTM C666 (Procedure B)  using twThe performance of geopolymer concrete against impact load was measured according to ACI 544  at 28 daFor the structural performance of GC slabs under 4-point bending, three steel reinforced slabs were cast from concrete mixes GC, 20R, and 100L. The selected mixes for the reinforced slabs had similar compressive strength of 32 MPa and were made to compare the structural behavior of the proposed lightweight GC mixes with that of the control GC mix. The reinforced slabs were cast in wooden moulds and left for 48 h before de-moulding and were then cured for 28 days in a water bath. White paint and grids were applied on the slabs to facilitate observation of cracks, see In this research, eleven GC mixes were produced, tested, compared, and analyzed. In addition, the structural performance of GC slabs made of selected mixes was tested and compared. Concrete characteristics including fresh and hardened durability and mechanical properties were determined such as workability, compressive strength, indirect tensile strength, freeze and thaw effect, and impact resistance. The concrete workability was measured through the traditional slump test and the flow diameter test according to AS 1012.3.5 . The aveThe flow table test results did not show clear differences between the tested mixes such as those observed from the slump results. All mixes showed an average flow diameter of around 500 mm, as shown in The compressive strength was measured in this study at concrete age of 3, 7, and 28 days to compare the development of geopolymer concrete strength with different lightweight fine aggregates. The measured average compressive strength of geopolymer concrete with vermiculite as a partial replacement of sand showed scattered results regardless of the vermiculite content. At 20% vermiculite content, the compressive strength increased by 14% at 3 days and decreased by 5% at both 7 and 28 days. At 40% vermiculite content, the compressive strength decreased by 3% at 3 days, increased by 5% at 7 days, and decreased by 4% at 28 days. At 60% vermiculite content, the strength decreased by 7% at 3 days, increased by 4% at 7 days, and increased by 1% at 28 days. The scattering of compressive strength using vermiculite might be attributed to the relatively very light weight and highly compressible nature of vermiculite compared with sand. This could allow the dolomite stone as a heavy material in the mix to compress the vermiculite particles and reduce their size, hence creating a degree of instability in the mixtures . Based oThe performance of geopolymer concrete that utilized LECA as partial or full replacement of sand was different from what was observed when using rubber or vermiculite, in that it showed higher compressive strength than that of the GC mix at all ages and all replacement ratios. The average strength increase ranged between 22% and 44% at 3 days, 16% and 39% at 7 days, and 15% and 27% at 28 days. The higher compressive strength of LECA geopolymer concrete was attributed to the higher fineness modulus of LECA compared with sand. The LECA used had fineness modulus of 4.3 compared with 2.2 for the replaced sand. The higher the aggregate fineness modulus, the higher the compressive strength of concrete [The rubber particles used had similar fineness modulus to that of LECA (fineness modulus of rubber was 4.85). However, rubber showed an adverse effect on the 28 day compressive strength. This is mainly attributed to the difference in the rigidity of the aggregate particles. Rubber particles are flexible in nature which results in low adhesion at the rubber/binder interface, and this increases with the increase of rubber content and hence compressive strength decrease. On the other hand, LECA particles are relatively rigid and have porous surfaces, which results in good penetration of their surfaces by the mortar and hence high adhesion at the LECA/binder interface.The 28 day indirect tensile strength was determined and plotted in The tensile strength of vermiculite mixes correlated to the corresponding 28 day compressive strength, where the tensile strength increased with increasing vermiculite content. All measured indirect tensile strengths of vermiculite mixes were higher than that of the control GC mix by 13%, 16%, and 18% at 20%, 40%, and 60% vermiculite content, respectively. This might be attributed to the deformability of the vermiculite particles that could reduce the crack initiation and propagation in geopolymer concrete under tensile loading and hence lead to higher tensile strength. Similar to the compressive strength pattern of LECA geopolymer concrete mixes, their indirect tensile strength increased by a constant value of 8–11% up to 60% LECA content, then started to decrease by 4% at 100% LECA content. The reasons for LECA geopolymer concrete tensile strength increase then decrease were the same as those for the compressive strength, due to the strong correlation between them. 3 concrete cubes to freezing-thawing cycles [The durability of geopolymer concrete was measured by exposing 100 mmg cycles . This waThe geopolymer concrete impact resistance was measured for mixes GC, 60 R, 60 V, and 60 L through a drop weight impact test using ten discs from each mix . The resThe results of tested one-way slabs under 4-point bending are presented in u) to the displacement at peak strength (dp). Compared with the GC-slab, both the R-slab and the L-slab showed 14% lower du/dp ratio as the du/dp ratio decreased from 1.4 to 1.2. This generally indicates the lower ductility of lightweight geopolymer concrete in this study compared with the normal weight geopolymer concrete at a given compressive strength.The stiffness of both the R-slab and L-slab were less than the GC-slab throughout the test. This was due to the fact of easy cracking of geopolymer concrete containing lightweight materials. The R-slab showed less stiffness than the L-slab due to the flexibility that rubber contributed to concrete when partially replacing natural sand. In R-slab and L-slab specimens, the slabs were able to resist the load until initial cracks occurred at about 11 kN load and 1.2 mm mid-span deflection for R-slab and 14 kN load and 1.0 mm mid-span deflection for L-slab. This was followed by a reduction in slab stiffness and then the load kept increasing until the slab mid-span deflection approached 6.5 mm at a load of 52 kN for R-slab and the mid-span deflection approached 5.6 mm at a load of 54 kN for L-slab. At this stage, clear shear cracks started to appear and became significant at the east side of R-slab when the slab reached its peak load of 68.2 kN at mid-span deflection of 11.1 mm. Similarly, the shear cracks became significant at the west side of L-slab when the slab reached its peak load of 57.8 kN at mid-span deflection of 6.6 mm. Once the R-slab and L-slab reached their peak loads, rapid increase was observed in the east deflection of R-slab, see The area under the load-deflection curves of each slab specimen has been determined to represent the toughness, shown in The R-slab displayed east toughness close to its mid-span toughness. The L-slab performed similarly but for its west toughness. This was again related to the location of failure of each slab in which the slabs started to deflect more at their middles, but once the load approached their shear capacities, the deflection increased at one of the slab sides and hence reflected on the measured toughness. Replacing 20% of geopolymer concrete sand decreased its middle, east, and west toughness by 47%, 9%, and 47%, respectively. Full replacement of geopolymer concrete sand by LECA decreased its middle, east, and west toughness by 75%, 74%, and 60%, respectively. Although both types of sand replacements showed less toughness than the corresponding GC-slab, the R-slab exhibited better toughness than the L-slab. This is attributed to the flexibility rubber can add to the geopolymer concrete, which increased its strain capacities and hence led to better toughness. The calculated average toughness of both R-slab and L-slab were about 89% of their mid-span toughness, which is higher than that shown by the GC-slab. This was due to the relatively higher deflections that occurred at east or west locations of those slabs (R-slab and L-slab) compared with their middles, hence higher average toughness.From the results above, it can be concluded that the performance of lightweight geopolymer concrete in toughness is lower than the normal weight geopolymer concrete having similar compressive strength. Some additive to the lightweight geopolymer concrete to enhance its flexural performance might be recommended for future research and comparisons.Three LVDTs were used to measure the deflection of each slab at locations named East, Middle, and West as shown in i.Replacing concrete sand by lightweight materials showed a clear effect on the GC slump, but not on its flowability under vibration. This indicated the ability of the lightweight GC mixes to flow well under vibration.ii.GC compressive strength increased when using 20% rubber content but decreased beyond that. Regardless of the vermiculite content, it showed scattered effect on the GC compressive strength. LECA showed significant increase in GC compressive strength by up to 44%, 39%, and 27% at 3, 7, and 28 days, respectively.iii.There was no significant effect on the GC compressive strength when applying freezing-thawing cycles (up to 30 cycles) in this study. Regardless of the lightweight fine aggregate used, the impact resistance of GC mixes decreased when replacing 60% of the sand volume.iv.The slab strength and deflection capacities decreased by 9% and 30%, respectively when using rubber, and by 23% and 59%, respectively when using LECA, compared with the control GC slabv.Partial or full replacement of GC sand by lightweight fine aggregate adversely affected the shear resistance of concrete and caused early shear failure of slabs. In addition, it decreased the slab stiffness and increased the rate of post-peak load degradation.This research investigated the mechanical performance, durability and structural behavior of geopolymer concrete (GC) containing lightweight materials. Eleven GC mixes were produced, tested, compared, and analyzed. The variables in this study included the lightweight fine aggregate material , and the GC sand replacement ratio  by volume. The measured fresh and hardened concrete properties and durability characteristics were workability, compressive strength at several ages, tensile strength, freeze and thaw effect, and impact resistance. The structural behavior of GC was also investigated in reinforced slabs made of selected mixes and subjected to flexural loads. The key findings of this study can be summarized as follows:Of the tested mixes, GC mix with 60% LECA (mix 60L) showed better mechanical performance than that shown by other mixes in this study. This indicates the applicability of this type of lightweight GC in structures subjected to axial loads. However, rubber would be the best lightweight material to recommend in this study to partially replace GC sand to resist impact and flexural loads. It is recommended for future studies to test the proposed GC mixes for durability, including drying shrinkage, water absorption, surface abrasion, carbonation, and conducting microstructural analyses.


Bacillus cereus MG708176, an alkali-tolerant, calcite precipitating, endospore-forming strain was added as a bio-healing agent after its immobilization on wood ash units. A spore concentration of [1.3 × 107 spore/cm3] combined with 2.5% w/w urea was added to cement. Beams of 40 × 40 × 160 mm were used and tested for completely damaged mortar specimens after 7, 14, and 28 days of water treatment. Using wood ash bacterial mortars, totally destructed specimens were fully healed in all time intervals. Positive changes in concrete mechanical properties in bacterial wood ash treatment that were 24.7, 18.9, and 28.6% force for compressive, flexural, and tensile strengths more than control. The micro-images of the Scanning Electron Microscope (SEM) showed the dense concrete structure via calcite, Bacillafilla, and ettringite formation. Our results have shown improvements in the concrete healing efficiency and the mechanical concrete properties by filling the concrete cracks using a calcite-producing bacterium that is immobilized on wood ash units.Novel carrier units were evaluated for their bio-healing benefits in our study to increase the efficacy of concrete healing.  Microbial CaCO3 is an ecofriendly and cost-effective material with a large range of engineering applications that are promising3. Bacteria have a natural capability to heal minor damage on their bodies in a relatively short time, without external influences4. The natural concrete ability of self-healing by the swelling of the cement particles due to un-hydrated particles hydration could occur without adding healing agents6.Bio-healing is a process that is biologically developed by certain microorganisms to heal cracks in concrete structures suffering from cracking that causes deterioration and shorter service life7. The urease activity of BICP enables them for use in the fields of environmental studies8.The efficient ingredient of bacterial-induced calcite precipitation (BICP) is urea because urease is well-covered across bacteria. Urease activity leads to pH increase and generation of nitrogen, hydroxides, and bicarbonate9. Bacterial introduction into concrete without using carrier compounds dramatically reduces bacterial survival over time10. As a result, to improve the efficacy of the bio-healing process, researchers have used different carrier compounds to strengthen bacterial survival inside the concrete. It is beneficial to use these carrier compounds, not only for increasing bacterial survival chances but also increase the concrete tensile strength11.It is worth noting that during the hydration process for cement, the bacterial cells or spores may be exposed to a risk of damage12. In our study, a new calcite forming bacterial isolate was inserted into the matrix of concrete and the impacts of the wood ash carrier on the bacterial capacity for bio-healing were investigated. These findings are used to compare a direct bacterial spore inoculation and the bacterial spores’ immobilization on wood ash to select the best technique for the introduction of spores into the concrete. According to a survey of the literature, wood ash is the burning wood waste  and the most important components in wood ash are CaO, SiO2, Al2O3, Fe2O3, and MgO, which react with moisture to form bonding agents14. Wood ash is a freely available agricultural waste that increases the quality, microstructure, use, and strength of concrete15. As a result, using wood ash in concrete is preferable because unburned carbon in wood ashes affects the level of pozzolanic material in the ash14. The use of wood ash units as a bacterial carrier is a promising method because, although it is thought of as a waste, it enhances the mechanical properties of produced composites, resulting in better strength and stiffness mixtures16 and offering inexpensive raw materials for the immobilization process. Using wood ash units improves concrete characteristics and that makes us to introduce a new approach to use it as a bacterial carrier to protect bacteria from harsh conditions of concrete.An attempt to treat the bacterial survival problem is by using bacterial carriers as encapsulation and bacterial immobilization on different carriers. As cracks occur, under the crack-tip tension, the embedded spores in crack surfaces are drawn into the crack and germinate to achieve healing17. The procedure of the endospore staining was performed as shown by Mokhtar et al.18. Alkali solid medium was prepared by adding NaOH solution droplets until pH = 14 to nutrient agar (NA)  and the bacterial isolate was cultivated on its surface18. The urease test described by Christensen19 was made. CaCO3 precipitation test was made as described by Fujita et al.20.An endospore-forming bacterium was isolated and identified as previously described by Hemida and Reyad7 spore/cm3 of the entire concrete mixture18. The nutrients, including 40 g/L calcium chloride anhydrous, 65 g/L urea, and 2 g/L yeast extract were dissolved in sterilized tap water for preparing the bio-based concrete.For the bacterial spores harvesting, the bacterial isolate was cultured for 24 h in alkaline nutrient broth (D (+)-glucose, 1 g/L, peptone, 15 g/L, sodium chloride, 6 g/L, yeast extract, 3 g/L, and then adding NaOH solution droplets until pH 14). The amended spore concentration in samples was 1.3 × 10Wood ash units of 0.2 mm particle size were created and impregnated with a mixture of nutrients and bacterial spores. Sawdust was obtained from carpentry shops and burned in an electric oven at 570° C. SEM–EDX analysis was used to characterize the wood ash see . Spores 21, and American Society for Testing and Materials (ASTM) standards22. The study consisted of two consecutive steps. Firstly, designed three different mixture types (mortar specimens), and tested them under direct incorporation and one carrier method. The spore suspension was injected directly during the last stage of the concrete mixing process.The testing program was conducted following Egyptian Code Practice (ECP)Subsequently, only the best technique  was chosen for the introduction into a concrete mixture. Negative control samples of mortar were named “C1” in which no bacterial spores or wood ash components were added. Wood ash only without bacterial spores, termed as “C2” was considered a positive control. In ''T1” specimens, Bacteria have been directly inserted into concrete ingredients without using any protective carrier compounds. “T2” is designated for the integration with bacteria as defensive carriers through using the wood ash units. Table The removal of the specimens was taken place after 24 h casting and then cured with water. Standard molds of Beam for mortar specimens (40 × 40 × 160 mm) were used. Compression and self-healing were conducted at various curing times of 7, 14, and 28 days for hardened concrete. The compressive, flexural, and indirect tests of splitting tensile strength for hardened concrete after 28 days were conducted for concrete specimens. Cylinder molds of 100 × 200 mm, 100 mm cubic, 100 × 100 × 500 mm beam were used. Besides, to track microstructural changes due to calcite formation, samples were also subjected to a SEM. Bacterial calcite precipitation was scanned by using SEM micrographs in micro-cracks specimens. These micrographs were created using a Carl Zeiss sigma 500 VP. A stereomicroscope was used for mortar self-healing measurements.23. For the slump test, a cone-shaped metallic mold with internal dimensions of a 20 cm bottom diameter, a top diameter of 10 cm, and 30 cm in height. This mold is settled on a surface that is flat, horizontal, and non-absorbent. A fresh concrete sample test must be taken immediately after mixing with a pan blender and put in a three-layer cone mold; a 25 times compaction for each one of the layers was taken place by a standard tamping rod. The mold of the cone is automatically separated from the concrete by moving it slowly and carefully in a vertical direction. This allows the concrete to recede. The mean level between the height of the mold and the highest point of the subsided concrete shall be measured.For measuring the strength of fresh concrete, the slump test is widely used. It was achieved per ASTM C143After 28th days from casting, 100 × 100 mm cube specimens were subjected to ECP 203 compression testing. To conduct the test, a two thousand KN (ADR 2000) compression testing system was used. Specimens were positioned following the ISO 4012 standard requirements on a rigid bottom bearing block with a spherical bearing block attached to the compressive testing unit.2 per second. The maximum compressive strength (σ) was determined by the division of the peak load (P) by the cross-sectional area (A) of each specimen. Three cubes were examined at 28 days using the following formulae.The compression load was acted on the specimen with a rate of the range of the ECP specified 0.6 N/mm24. The load was adjusted to the ASTM standard at 900 kPa per minute, which is at the midpoint of appropriate load speeds. The test called for a plate strip to be placed on the top and the bottom of each specimen was used as a bearing strip, and the strips were applied for each test. The data collected for these tests included the load as recorded by the testing machine. This data was then recorded to calculate the T of each specimen by dividing twice the peak load (P) by the product of pi, the diameter (D), and the length of the cylinder (L). Three cylinders were examined at 28 days and their average value is reported by following Eq.  for the calculation of the rupture modulus which is assessed by the division of the product of peak load (P) and specimen clear span (L) by the product of specimen width (b) and depth (d) squared.The static flexural test was made 28 days after the casting of the specimens. This test was conducted in conjunction with the ASTM C78 standard26. The level of significance in all tests was P ≤ 0.05. The results are expressed as means ± standard error (SE).Data were statistically analyzed using a two-way analysis of variance (ANOVA test) using SPSS Statistical Package Program version 23. Means of treatments were compared by Duncan multiple range test when the differences were significantThis article does not contain any studies with human participants or animal.3white powder immediately appeared in the media after the bacterial inoculation and its density reached a high level 7 days after incubation Table 17 as Bacillus cereus using 16SrRNA gene sequencing and its sequence had an accession number.It is observed that the synthesis of endospores with bacterial bacilli forms, the positive result for urease test was recorded after 16 h of incubation, and a calcium carbonate powder was observed in NB appended with urea and calcium chloride (NB-U/Ca). A CaCOThe stereomicroscopic images in Figs. For the detection of flexural intensity, Samples with bacterial wood ash for the incorporation showed maximum strength and improvements of 22.22, 13.63, and 14.29% in flexural strength at days 7, 14, and 28 compared negative control specimens  was detected (Fig. Calcite and ettringite were observed in bio-concrete cracks as shown in Fig. ted Fig.  in the bThe slump estimation value of the normal concrete was found to be 30 mm in this sample. The slumping value of bio-concrete is recorded to be 40 mm. Table 28. Our findings showed the decreased crack healing of direct inoculation particularly after 28 days may be due to a decline in the feasibility of bacteria survival inside concrete and the loss of bacteria by removal just for the construction of dense microstructures formed11. Specimens integrated with wood ash carrier units displayed the greatest healing as a consequence of bacterial immobilization and protection from harsh conditions that enable spores persist very long periods29.This study aims to assess the reflection of wood ash units as a bacterial carrier on concrete self-bio-healing efficiency and mechanical properties. Our results revealed that all bacterial integration specimens result in increased bio-healing efficiency more than control and that verified by micro-graphs from the SEM that showed reaction product morphology, which was calcium carbonate and ettringite needle-like phase and is similar to that observed by the cement that was hydrated3 on the microorganism cell surfaces and inside mortar pores3. As cracks occurred, activation of the incorporated bacterial spores, and CaCO3 is formed by a supplied nutrients metabolism30. This calcium carbonate continuously manufactured by the bacteria, urea, and calcium chloride provided as organic precursors makes the internal structure of concrete more compact.Our study showed that all methods of bacterial integration contribute to increased flexural strength of the mixtures, due to this deposition of CaCO35 confirm that the bio-healing is a cause of compressive strength improvements in opposition to normal gross aggregates, these improvements may be related to a smaller wood ash units scale. This permitted better packing and the concrete matrix's compaction around them, which gave these specimens much more strength than control specimens10. An explanation of the increase in concrete strengths is due to the synergistic effect of wood ash units and calcium carbonate-producing bacteria.The rise in compressive strength is in agreement with the results obtained by16 and providing cheap raw material for immobilization process.Using wood ash units as a bacterial carrier is a promising material where it is considered a waste that improves the mechanical properties of produced composites leading to better strength and stiffness mixtures3 using a calcite-producing bacterium. Our work offers a low-cost raw material for bacterial immobilization as wood ash is regarded a waste and can be used as a potential bacterial carrier that enhances the mechanical characteristics of generated composites.Our results suggested that wood ash works as an efficient immobilization technique to protect bacterial cells from harsh conditions in the concrete. The effective positive role of the immobilized bacteria in the improvement of compressive, tensile, and flexural strengths of concrete was detected by filling the concrete cracks with CaCOSupplementary Information.


In order to study the protection performance of silane coating on in-service concrete structures in a sulfate environment, we collect concrete samples in the field to simulate the concrete erosion process by accelerated erosion with wetting–drying cycles. We place the samples into protected, exposed and control groups corresponding to a corrosive environment with silane protection, corrosive environment without protection and general environment for three different service conditions. A combination of ultrasonic velocimetry, CT (Computed Tomography) scan imaging, NMR (Nuclear Magnetic Resonance) pore structure analysis, strength testing and other methods are used to analyze the strength, ultrasonic wave velocity, pore structure and other characteristics of the specimens during sulfate erosion. Based on the test results, the protective effect of silane coating on concrete structures under sulfate attack is quantitatively analyzed, and an index for judging the damage rate of specimens is proposed to quantitatively analyze the protective effect of silane coating. The research results show that the damage of the concrete structure under silane protection in a sulfate-attack environment can be reduced by more than 50%; its integrity damage index and strength damage index are easily affected by the location of local defects, which leads to a decrease in the protection efficiency of the surface silane coating. In the last three decades, bolstered by large-scale infrastructure construction, a huge amount of concrete structures in service have existed in China. In the western region of China, there are a large number of salt lakes, caves and deposits rich in sulfate components which are in contact with concrete bodies using water as a medium. This causes more serious damage to the durability of concrete structures through combined internal–external erosion and physical–chemical corrosion ,2,3,4,5.Usually, sulfate enters the concrete interior by penetration using water as a medium. It leads to structural deterioration through mechanical or chemical interaction with the cement paste ,15,16, wTo counteract sulfate damage to concrete structures, two methods are usually used to enhance the erosion resistance of concrete. One method is to adjust the ratio of concrete. By adding additives and improving cement varieties, the structure and environment inside concrete can be adjusted so as to inhibit the production of corrosion products . This apThe prediction of the service-life of concrete impregnated with silane by means of field statistics combined with probabilistic models can be a feasible way to judge the protection effect ,28. HoweExisting studies have shown that silane protection has a positive effect on the prevention of sulfate attack as an effective protective measure. However, silane-coating protection mainly depends on the hydrophobic layer generated on the concrete surface, and the surface integrity directly determines its protection effect. According to the requirements of JTJ275-2019, the thickness of a silane protection layer is mostly 2–4 mm. In the actual service process, if the protection layer is damaged by external collision, stress cracks or other factors, local protection failure may occur. Referring to the study of service tunnels in the literature ,41, to cThe specimens selected for this experiment are from a river-crossing tube sheet in service. The design strength grade is C50, the 28-day compressive strength is 61.6 MPa and the water–cement ratio is 0.4, according to the relevant research ,43 and tTo prepare the specimens, the poured pipe pieces were first broken using a crushing hammer to extract the reinforcement inside the pipe pieces. A concrete core sample with a diameter of 50 mm was extracted using core-drilling equipment. Then, the core sample was processed into a 50 mm × 100 mm cylindrical specimen. The specimens were prepared for testing, with an age of 1150 d and an original compressive strength of 75 MPa.2. The surface area of the specimens used in this paper is about 0.01963 m2, and the corresponding design amount is about 8.8357 g. Considering the accuracy required for actual operation and measurement, the actual amount of silane used in this paper is 8.9 g for each specimen. The specimens were divided into a protected group, exposed group and controlled group. The protected group was coated with the designated amount of silane before the experiment, and the amount used per single specimen was 8.9 g. The exposed group and the controlled group were left untreated.The original tunnel used isooctyltrioxethylsilane silane to spray a protective coating on the surface of the tube sheet before it was put into use. The experimental specimens used in this paper restore the use of silane materials on site, and the designed amount of silane for concrete in the reference project is 450 g/mThe wetting–drying experimental maintenance cycle consists of an immersion phase (wet phase) and a drying phase (dry phase). The experimental immersion phase simulates the erosion process of the tunnel-tube sheets by immersing the test blocks in the erosion solution of the corresponding composition. During the immersion period, the test blocks of the protected and exposed groups were placed upright in a PVC box with a lid containing a 10% mass fraction of sodium sulfate solution. The controlled group was placed in the same style of PVC box with water. The top of the specimen was placed in the PVC box with the same water, the distance between the top of the specimen and the liquid level in the box was not less than 3 cm and the solution in the box was changed once every 30 days. The set maintenance temperature was 20 ± 0.5 °C, and the relative humidity was 95 ± 0.5%. The specimens entered the drying stage after the specified soaking time. The specimens were taken out from the erosion liquid box, the residual liquid on their surface wiped off and then placed in a blast-drying box. The specimens were dried at 60 °C for 6 h and placed in a cool and ventilated place for 2 h, then put back into the original erosion solution box for the next wetting–drying cycle. Three groups of specimens started the cycle at the same time, and the number of cycles for each group of specimens was 180 times. Preparation and curing of specimens are shown in The specimen grouping and quantity design are shown in In this paper, three types of methods are combined to analyze the changes in the properties of concrete specimens during rapid wetting–drying cycles of erosion. Ultrasonic testing was used to analyze the overall condition of the concrete. The structural changes to the specimens were analyzed using CT scanning and NMR testing. Strength tests were used to compare the strength changes of the specimens under different conditions. The overall test protocol is shown in A non-metallic ultrasonic tester was used to test the longitudinal wave velocity on the side of concrete specimens at a sampling rate of 0.05 μs. After every ten wetting–drying cycles, a test was conducted on the specimens of the protected group, the exposed group and the controlled group. The test specimens were divided into four test planes at equal intervals of 20 mm from top to bottom. Nine measurement lines are laid out at equal intervals at 20° angle for each measurement surface. The longitudinal wave velocity of each specimen was tested at 36 points in total, and the test situation is shown in The specimens’ structure was analyzed using an industrial CT system. We use the model Y.CT.Modular X-CT scanning equipment, with continuous spiral scanning mode, tube voltage 225/450 KV, imaging plate size 400 mm × 400 mm, imaging plate pixel dot size 0.2 mm, detectable diameter range 0~800 mm and maximum penetration thickness 60 mm Fe. The specimens were scanned in high precision slices for the protected and exposed groups at the end of 60, 120 and 180 wetting–drying cycles. The minimum scanning resolution was 5.3 μm, and the number of slices scanned for a single specimen was 1600.After the CT tests were completed for the protected and exposed groups, we used a Suzhou Newmark AniMR-150 NMR instrument to perform NMR tests on the specimens before and after the erosion experiment to obtain pore data. The magnetic field strength of the test system is 0.3 ± 0.05 T, the peak output is not less than 300 W and the linear distortion is less than 0.5%. The specimens were soaked in water for 48 h to saturate the specimens before the test was started.d is damaged was recorded. The uniaxial compressive strength can be calculated according to the following formula.The specimens of the exposed group and the specimens of the protected group that completed the NMR test were dried at 60 °C to the state before immersion. The specimens of the exposed group, protected group and controlled group were uniaxially loaded at 0.5 MPa/s using a servo material-testing machine, and the peak load when the specimen diameter Finally, the overall structure of this paper is shown in (1) AppearanceThe surface of the exposed group specimens showed a white powdery substance at some locations on the surface before cleaning. Its composition was tested by XRD (X-ray Diffraction) and found to be mainly sodium sulfate, mostly distributed at the joints between the aggregate and the cement paste. After cleaning the surface, it was found that significant cracks were produced in the area where the white powder appeared. This indicates that these cracks are mainly due to the physical salt erosion caused by the heating of the sodium sulfate erosion solution inside the specimen during the drying process.(2) Ultrasonic wave speedThe specimens in this paper were made directly from in-service structural concrete, which has a large structural variability. This is also reflected in the variation of the wave velocity during the test period. Three typical patterns of variation at different measurement points of the exposed group specimens are shown in From the rate-of-change of wave velocity in Differing from the exposed group test blocks, the protected group test blocks basically showed two trends of variation. Three test points were selected in (3) StructureIt can be seen in (4) StrengthThe uniaxial compressive strengths of each group are shown in Unlike other erosion-damage processes, sulfate erosion of concrete is a process of strengthening before damage. Therefore, within the erosion process, the area where the concrete is in contact with the erosion medium will have some of its pores filled first. Along with the subsequent erosion process, the concrete structure begins to deteriorate, and new pore structures are formed one after another.If the porosity Since the newly generated pores must be caused by the material damage resulting from erosion, then the equivalent volume amage is :(3)dp=ΔpBased on the above idea, we can calculate the The calculations in In this paper, based on the uniaxial compressive strength results of the specimens, the strength-loss of the material is calculated by the following equation:Based on the strength test results above, we can conclude that the strength damage for the exposed group is 0.274 and for the protected group is 0.174.In order to improve the accuracy of ultrasonic wave velocity testing, this paper divides the specimen into four equally spaced measurement surfaces Dynamic modulus tionship :(5)Ed=(1Let In the concrete sulfate erosion experiment, the composition As a result, the measurement surface is divided by the center of the measurement line, and the longitudinal wave velocity measured by the measurement line represents the area where the measurement line is located. The equivalent kinetic mode Using the equivalent dynamic modulus as the damage factor, the initial equivalent dynamic modulus of the specimen is Damage degree From the calculation results in Therefore, this paper considers that the damage degree of the specimen should be taken as the maximum value of the damage degree of the four measurement surfaces.When comparing and verifying the protection effect of specimens in the engineering field, due to the variability between specimens, the protection effect can be judged by comparing the damage parameters The From the results in However, in the actual experiments, the specimens prepared in this paper are from actual structures. There are some differences in the homogeneity of the prepared specimens.The results obtained from the experiments showed that the overall structure of the concrete was effectively protected by the silane coating. During the accelerated erosion tests conducted, the concrete specimen exterior and the concrete pore structure were maintained in a more stable state. The use of silane protection is undoubtedly positive and effective in safeguarding the health of concrete structures.However, the results of the intensity and wave velocity tests were compared between the protected and exposed groups. It is easy to find that the wave velocity damage and strength damage of the silane protection specimens are smaller than that of the exposed group specimens. However, the protection efficiency in terms of strength damage and wave velocity damage is much less than that in terms of structural protection. The reason for this is that more attention was paid to the weak areas when considering the wave velocity and strength damage of the specimens. Since sulfate erosion is a result of a combination of physical damage and chemical reaction, when other areas are better protected, corrosion products at the weak point may accumulate rapidly, thus further amplifying the corrosion deterioration caused by local damage. In this paper, the raw materials used in the preparation of the style samples are from actual structures, and there are some differences in their uniformity when prepared as small-size samples. Some of the raw defects lead to a situation when the protective coating is set with silane where it is easy to miss parts and undercoat. In addition, the good water-barrier effect of silane, contrary to expectation, makes the parts with micro-defects on the surface after protection more quickly destroyed by crystallization.In this paper, the corresponding industry specifications and similar studies were referred to when designing the test procedure ,46. A scAt present, the work on durability protection of concrete structures under conditions of sulfate erosion is still in the exploratory stage, and many structures are still in the early stage of service, lacking corresponding research accumulation. In this paper, we try to simulate the erosion process in an actual environment by accelerated erosion under the premise of field sampling. At the same time, a diversified detection system is proposed according to the key indicators of concrete erosion deterioration processes. Of course, there is still development of the corresponding detection techniques used in this paper and the subsequent data processing and analysis techniques. Through waveform recognition in ultrasonic velocimetry and image-recognition processing technology in CT inspection, the optimization of these technologies can help further the accurate evaluation of damage results. At the same time, the protection efficiency index based on damage factors proposed in this paper is still an index-pointing judging method. It is necessary to develop a systematic evaluation and analysis method to form a comprehensive judgment on the effectiveness of protection measures.Various testing methods were used to judge the damage of concrete within a sulfate erosion environment. The main research results include:1. An ultrasonic test method with multi-point determination of sound velocity was proposed to determine the characteristics of sound velocity variation during erosion deterioration. The test results demonstrate that the field-prepared specimens showed significant differences in wave velocity variation during the erosion deterioration process according to the material differences at the interface of the measurement points.2. Sulfate erosion-acceleration experiments were carried out with actual engineering samples. The acoustic, structural and strength degradation patterns of the protected group and the exposed group under the influence of sulfate erosion were compared. It is shown that the specimens in the protected group still maintain a high overall structural integrity in the erosion test. However, there was also a more-significant loss of their compressive strength.3. A critical index of protection efficiency based on the variation of the damage factor was proposed. The protective measures adopted in the subject tunnels were analyzed by using the critical index for coated silane protection. This type of measure has excellent effects in protecting the overall structure of the specimen. However, it is not effective for local damage or weak areas. The integrity and uniformity of the protection layer should be ensured as much as possible when used, and the existing defects should be repaired in time.


Viridovipera and is sister to Trimeresurus medoensis based on molecular data for mitochondrial cytochrome b gene, whereas resembles Trimeresurus gumprechti morphologically. A combination of characters helps delimit the new species from its congeners. Description of the new species highlights the need for dedicated surveys across northeast India to document its reptilian diversity, as this represents the third new species of the genus to be described in the past three years.A new cryptic species of green pit viper is described from northeast India, based on specimens collected from the state of Mizoram and Meghalaya. The new species is a member of the subgenus  Trimeresurus Lacépède 1804 currently comprises 43 nominate species [T. albolabris Gray, 1842, T. arunachalensis Captain, Deepak, Pandit, Bhatt & Athreya, 2019 T. erythrurus , T. gumprechti, David, Vogel, Pauwels & Vidal, 2002, T. medoensis Zhao, 1977, T. popeorum Smith, 1937, T. salazar Mirza, Bhosale, Phansalkar, Sawant Gowande, Patel 2020, T. septentrionalis Kramer, 1977, and T. yunnanensis Schmidt, 1925, are recorded from Northeast India [Trimeresurus often result in misidentification [T. albolabris from several localities across the region [T. salazar as highlighted by Rathee et al. [The genus fication . For exae region  most like et al.  & Malik e et al. .T. gumprechti  can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix “cyt b) gene was amplified using primers used by Pyron et al. [Trimeresurus spp. available on GenBank® were downloaded for calculating genetic divergence and/or molecular phylogenetic reconstructions. Taxa for molecular phylogenetics through a Maximum Likelihood approach, were selected based on the tree topologies recovered by Mirza et al. [http://iqtree.cibiv.univie.ac.at/) online portal [th generation. The analysis was terminated after the run reached a split frequency below 0.01 and convergence was assessed by checking if the ESS values were greater than 200 for all parameters using Tracer v1.6. First twenty-five percent of the trees were discarded as burn-in. Uncorrected pairwise p-distance (% sequence divergence) was calculated in MegaX with pairwise deletions of missing data and gaps.Genomic DNA was isolated from the preserved tissues of the type specimens using QIAGEN DNeasy kits, following protocols directed by the manufacturer. Molecular methods follow Mirza et al.  and Mirzn et al.  and Mirzn et al. . A 22.4 a et al.  using Cla et al.  with defe portal . Sequence portal . Generalcyt b gene for members of the subgenus Viridovipera and Trimeresurus macrops was used as an outgroup for the analysis. The ML analysis recovered two clades, one containing Trimeresurus medoensis and sequences of Trimeresurus from Mizoram and Meghalaya; the second clade contains Trimeresurus gumprechti, Trimeresurus stejnegeri, Trimeresurus truongsinensis and Trimeresurus vogeli . The BI analysis resulted in a slightly different tree topology with polytomy. Trimeresurus gumprechti was recovered monophyletic in BI .The molecular phylogeny was based on 1080bp of s vogeli . Relatioic in BI  and paraic in BI  employedTrimeresurus stejnegeri in part Malhotra & Thorpe 2004: 230Trimeresurus yunnanensis in part Malhotra & Thorpe 2004: 230Trimeresurus gumprechti David & Mathew 2005: 87urn:lsid:zoobank.org:act: 8E1EF11A-0488-4AC0-B0FC-DE95E838FB15Figs Table 1Adult male NCBS NRC-AA-0012 (ex. MZMU 2043) from the ground in the homestead garden , Bethel Veng, Champhai, Champhai District, Mizoram. Collected on 24 October 2020 by P.S. Muanga, H.T. Lalremsanga, Lal Muansanga and Lal Biakzuala.Adult males, BNHS 3658 & BNHS 3660; two adult males, VR/ERS/ZSI/834 MZMU2730 (field number YRS005) and VR/ERS/ZSI/835 (field number YRS006), two adult female VR/ERS/ZSI/832 (field number YRS001) and VR/ERS/ZSI/833 collected from near a residential area, Umroi Military Station, Umroi, Ri-Bhoi District, Meghalaya . Collected in the last two weeks of February 2021 by Yashpal Singh Rathee.ZSI/ERS 486 & BNHS 2634, Shillong, Meghalaya, India.Trimeresurus, characterized by (1) hemipenes short and strongly spinose; (2) body green in both males and females; (3) interstitial skin black; (4) moderate size, with maximum total length of 750mm; (5) conspicuous bicolored postocular stripe in males, thin and white below, wide and bright red above, faint white or no postocular stripe present in female (6) vivid, wide bicolored ventrolateral stripe, deep red below/white above in males, extending along the lower half of the tail, white in females; (7) eyes rust coloured in males, green in females; (8) tail mostly rusty or reddish-brown (9) V: 157–162; SC: 54–67 in males and V: 153; SC: 54–55 in females; (10) first supralabial distinct from nasal; (11) 19 or 21 dorsal scale rows at midbody, moderately keeled; (12) snout covered with rather enlarged juxtaposed scales; (13) internasals never in contact, separated by 1–2 scale; (14) supraoculars narrower than internasals, separated by 9–10 smooth cephalic scales.A species of the genus The species epithet is an eponym honouring late Maya Singh Rathee, mother of Yashpal Singh Rathee. English name: Maya’s pit viper.th to 80th ventral ; 3rd supralabial is the widest amongst all supralabials (4 mm) and is in contact with 2nd and 4th supralabials, lower preocular and a small scale between subocular and supralabials; 4th supralabial small, separated from the subocular by a single row of smooth scales; 5th supralabial in contact with temporal; the remaining supralabials slightly decreasing in size posteriorly and in contact with temporal scales; 10/11 infralabials, the first pair in contact with each other; the first three pairs in contact with anterior chin shields; six pairs of chin shields, 1st, 2nd, 5th and 6th contact medially, 3rd and 4th separated by a pair and a single small scale respectively; separated from infalabials by 1–5 scale rows  coloured in T. stejnegeri Schmidt, yellow or yellowish green in T. vogeli David, Vidal & Pauwels, green or yellowish green in T. medoensis Zhao, and bright or deep red in T. yunnanensis, and a green coloured eye in females versus yellow or amber in T. stejnegeri, yellow in T. vogeli and golden yellow in T. yunnanensis. A bicoloured (broad red above and thin white below) postocular streak is present in males of Trimeresurus mayaaesp. nov. versus white in T. stejnegeri, white, thin and faint in T. vogeli, no postocular streak in T. yunnanensis. A white ventrolateral stripe is present in female Trimeresurus mayaaesp. nov. versus red + white in T. medoensis and pale green in T. yunnanensis. Trimeresurus mayaaesp. nov. has 19–21 dorsal scale rows at the midbody versus 17 in T. medoensis and 19 in T. yunnanensis. Trimeresurus mayaaesp. nov. with 3–4 palatine teeth versus 5 in T. yunnanensis; Trimeresurus mayaaesp. nov. with 10 pterygoid teeth versus 12–14 in T. yunnanensis & T. stejnegeri. The new species differs from members of Viridovipera in showing a pairwise sequence divergence (p-distance) of 5–10% for cytochrome b gene . Trimeresurus mayaaesp. nov. has 19–21 mid-dorsal scale rows versus 17 in T. arunachalensis; 23–25 in T. erythrurus.Trimeresurus mayaaesp. nov. differs from all the other species of Trimeresurus by a combination of following characteristics:Trimeresurus mayaaesp. nov. has a short spinose hemipenis versus a long papillose hemipenis without spines in T. fasciatus (Boulenger), T. flavomaculatus (Gray), T. gunaleni Vogel, David & Sidik. T. guoi Chen, Shi, Vogel & Ding, T. insularis Kramer, T. kanburiensis Smith, T. macrops Kramer, T. mcgregori Taylor, T. nebularis Vogel, David & Pauwels, T. phuketensis Sumontha, Kunya, Pauwels, Nitikul & Punnadee, Trimeresurus sabahi Regenass & Kramer, T. sumatranus (Raffles), T. tibetanus Huang, T. venustus Vogel, T. yingjiangensis Chen, Zhang, Shi, Tang, Guo, Song & Ding.Trimeresurus mayaaesp. nov. has 19–21 mid-dorsal scale rows versus greater than 23 scale rows in T. andersonii Theobald, T. cantori (Blyth), T. gracilis Oshima, T. purpureomaculatus (Gray) and less than 19 scale rows in T. kanburiensis, T. macrolepis Beddome, T. malcolmi Loveridge, T. trigonocephalus (Latreille).Trimeresurus mayaaesp. nov. first supralabial is completely free from nasal versus first supralabial partially or completely fused with nasal in T. andersonii, T. cantor, T. cardamomensis , T. davidi Chandramouli, Campbell & Vogel, T. hageni (Lidth De Jeude), T. labialis (Steindachner), T. purpureomaculatus, T. rubeus In Trimeresurus mayaaesp. nov. has an immaculate green dorsal colouration versus dorsal colouration with blotches, spots, bands or crossbars in T. andalasensis David, Vogel, Vijayakumar & Vidal, T. andersonii, T. borneensis (Peters), T. brongersmai Hoge, T. cantori, T. fasciatus, T. flavomaculatus, T. gracilis, T. gramineus (Shaw), T. gunaleni, T. guoi, T. hageni, T. honsonensis , T. kanburiensis, T. labialis, T. malabaricus (Jerdon), T. mcgregori, T. mutabilis Stoliczka, T. puniceus (Boie), T. purpureomaculatus, T. schultzei Griffin, T. strigatus Gray, T. sumatranus, T. tibetanus, T. trigonocephalus, T. truongsonensis Orlov, Ryabov, Thanh & Cuc, T. venustus, T. wiroti Trutnau.Trimeresurus mayaaesp. nov. has a bicoloured (red above white below) postorbital stripe versus postorbital stripe absent in T. caudornatus Chen, Ding, Vogel & Shi and T. sichuanensis (Guo & Wang).Emblica officinalis, Mangifera indica, Psidium guajava, Prunus domesticus, etc. at Bethel Veng, Champhai, a district capital of Champhai District, Mizoram. According to Champion and Seth [9/C2) which is mostly dominated by Pinus kesiya with ether associates like Quercus spp., Schima wallichii, Rhododendron spp., Castanopsis spp., Lyonia ovalifolia, Rhus spp., Myrica esculenta, Prunus spp. etc. Champhai has a moderate climate. In winter, the temperature varies from 10°C to 20°C and between 15°C and 30°C in summer, with 2037.6 mm annual rainfall. All the paratypes were found during early hours of night in a forested patch next to a stream within Umroi Military Cantonment area. An individual was spotted on the ground, crossing a track. In captivity, the specimens fed on the Rhacophorus bipunctatus, Leptobrachium sp. and Minervarya sp.The holotype was collected at dusk at around 17:15h. The specimen was found on the ground in the homestead garden planted with and Seth , the arecyt b suggests that the new species is a member of the subgenus Viridovipera. Based on 1080bp of cyt b gene, the new species is sister to T. medoensis s. l.. Further work with data for additional genes is necessary to resolve the phylogenetic relationship of the new species with other members of the subgenus because of unresolved relationships in BI. The new species shows an intraspecific divergence of 0–4% and an interspecific divergence of 5.2–10% from members of the subgenus Viridovipera  suggests that the records of T. yunnanensis, T. stejnegeri and T. gumprechti represent the new species described herein.Molecular data for green pit vipers from throughout their range hints on presence of morphologically cryptic species –33. Lackost taxa . Similarost taxa  identifid Mathew  considerst India  to be ofTrimeresurus from across India would be necessary to ascertain the exact diversity within the genus and its biogeography. Molecular data for T. medoensis s. l. suggests presence of distinct lineages across sampled areas of Indo-China, so does T. gumprechti. Sampling for pit vipers from across Northeast India, especially of widespread species like Trimeresurus popeiorum, Trimeresurus albolabris and Trimeresurus erythrurus, will help evaluate if there are cryptic species within broadly distributed and morphologically cryptic groups. Northeast India is rich in its reptile diversity, which still remains poorly studied. Several new species have been described in the recent past [Recent investigations on the systematics of pit vipers of northeast India have led to the discovery of three new species, including the present work , 34. An ent past . Many roS1 FigNumbers at nodes indicate ML bootstrap support through an ultra-fast search method.(PDF)Click here for additional data file.S2 FigNumbers at notes indicate BI posterior probability.(PDF)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


The occurrence of antibiotics in the environment could result in the development of antibiotic-resistant bacteria, which could result in a public health crisis. The occurrence of 676 antibiotics and the main transformation products (TPs) was investigated in the 48 wastewater treatment plants (WWTPs) from 11 countries  by target and suspect screening. Target screening involved the investigation of antibiotics with reference standards (40 antibiotics). Suspect screening covered 676 antibiotics retrieved from the NORMAN Substance Database (antibiotic list on NORMAN network). Forty-seven antibiotics were detected in effluent wastewater samples: thirty-two by target screening and fifteen additional ones by suspect screening. An ecotoxicological risk assessment was performed based on occurrence data and predicted no effect concentration (PNEC), which involved the derivation of frequency of appearance (FoA), frequency of PNEC exceedance (FoE), and extent of PNEC exceedance (EoE). Azithromycin, erythromycin, clarithromycin, ofloxacin, and ciprofloxacin were prioritized as the calculated risk score was above 1. The median of antibiotics’ load to freshwater ecosystems was 0.59 g/day/WWTP. The detection of antibiotics across countries indicates the presence of antibiotics in the ecosystems of Europe, which may trigger unwanted responses from the ecosystem, including antibiotic resistance. Antibiotics have been extensively used by human in various ways; on top of medical use, they are also employed in agriculture, aquaculture, animal husbandry, horticulture, food preservation, and industries such as ethanol production . This enUnder exposure to antibiotics, bacteria which manage to survive would be able to proliferate and to pass the resistant traits to succeeding generations . AntibioWithout proper wastewater treatment, the disposal of antibiotics could lead to the development of bacteria that is not susceptible to antibiotics, superbugs, which could result in serious public health crisis followed by thousands of deaths ,11. In lAn extensive literature review was conducted to select a set of antibiotics to be studied in effluent wastewater samples, which is available to the scientific community on the NORMAN Suspect List Exchange as the List S6 ITNANTIBIOTIC . A smallThe aims of the study were (1) to screen for antibiotics in the effluent wastewater samples with a special focus on the less studied ones; (2) to establish the occurrence profile of antibiotics across European countries; and (3) to perform ecotoxicological risk assessment and prioritization of the detected antibiotics. The ultimate goal of this study was to investigate the emission profile of antibiotics from WWTPs in Europe.Target screening identified 32 antibiotics in the wastewater. Five of them were prioritized as they showed risk score above 1: three macrolides  and two quinolones (ofloxacin and ciprofloxacin). Some of the prioritized antibiotics of environmental concern have been characterized in previous studies. Azithromycin and ciprofloxacin were proposed as markers of antibiotic pollution for the analysis of effluent wastewater . ClarithWide-scope suspect screening of antibiotics was performed to avoid overlooking analytes, which successfully detected 15 antibiotics overlooked by target screening. Most of these compounds were not screened for by the conventional target screening approach in previous studies of antibiotics in WWTP effluents ,19,20. TAzithromycin is one of the most commonly used antibiotics and is frequently found in WWTP owing to its low metabolic rate  and longThe common sewage treatment process was not able to remove erythromycin and its main degradation products  from recHelicobacter pylori [Clarithromycin in wastewater posed a risk as it would aid the development of resistance in multiple bacteria including r pylori . A novelr pylori  was ableOfloxacin and ciprofloxacin both pose significant environmental risk due to their genotoxicity ,30, and Despite having a risk score below 1, some antibiotics were detected in over half of the wastewater samples studied, including sulfapyridine, trimethoprim, lincomycin, flumequine, metronidazole, sulfamethoxazole, oxolinic acid, and amoxicillin by target screening and 8-hydroxyquinoline, clindamycin, linezolid, fluconazole, sulfasalazine, mycophenolic acid, roxithromycin, and cordycepin by suspect screening. Target screening of these suspected antibiotics would enhance the robustness of the concentration determination, which is essential for checking for PNEC exceedance. The majority of these antibiotics were detected below P-PNEC. The frequency of PNEC exceedance (FoE) of these antibiotics was between 0 and 0.01, which resulted in a risk score below 1. The derivation of robust EQS PNEC values is important to verify that these antibiotics at detected levels do not pose threats to the ecosystem.Antibiotics are commonly used together in synergistic antimicrobial combination  for treating bacterial or fungal infections. A common pair is trimethoprim and sulfamethoxazole , which wTotal antibiotic load per WWTP spanned from below 1 g/day to over 7000 g/day, which was a result of the broad range of scale of the WWTPs—small WWTPs such as the ones in Bremen–Seehausen and Bergheim serve less than 10,000 people, which resulted in less than 1 g/day of total antibiotics load, while large WWTPs such as the Athens WWTP serves over 3.7 million people, which resulted in 7000 g/day of total antibiotics load. The majority of the small WWTPs would load the effluent wastewater (together with antibiotics) to river water ecosystems, while the large WWTPs such as the one in Athens would load the effluent water with antibiotics to sea water ecosystems, which gives a much higher dilution factor to the final concentration of antibiotics that exist in the destination. Therefore, it would be misleading to compare just the total antibiotic load between WWTPs without considering the flow rates and the disposal ecosystem.Germany showed a general total antibiotic load profile of below 100 g/day in the majority of WWTPs, which was an order of magnitude lower than WWTPs of similar flow rates across DRB countries such as the ones in Varazdin, Ljubljana, and Zilina. This fact suggested a possible better performance in antibiotic removal for the German WWTPs. Sabac was among the top three WWTPs on daily loads, which is consistent with previous finding of high antibiotic release at the area due to the local pharmaceutical industry  . NeverthGerman WWTPs showed a similar pattern in which erythromycin accounted for 14–60% of the loads in 28 out of 35 of the German WWTPs studied. This further proves the need for proper removal for erythromycin (a prioritized antibiotic) in wastewater, especially in Germany. Erythromycin, azithromycin, clarithromycin, sulfadiazine, and trimethoprim revealed loads of > 1 kg/day across the 48 WWTPs. These antibiotics can potentially be used as additional indicators when optimizing the wastewater performance. The median total load (sum of loads in 48 WWTPs for a given antibiotic) was 35.9 g/day, while the median individual load per WWTP for a given antibiotic was 0.59 g/day. Advanced post treatment following the CAS system (adopted in majority of the studied WWTPs) could significantly enhance the removal of antibiotics, namely UV and ozonation .Despite the high amounts of antibiotics entering freshwater ecosystems through effluent wastewater discharge which could accelerate the evolution of bacteria into superbugs, there is not yet a regulatory system to control the environmental risk caused by these compounds. Certain methodologies had been proposed for setting the regulatory limits based on PNECs, including the derivation of microbial system-specific minimal selective concentrations (MSCs) ,43, and Effluent wastewater samples were collected from various projects from August 2017 to October 2018 ,46, and After collection, samples were stored in the WWTP at −20 °C and remained frozen during transportation. Effluent water samples were cleaned up and pre-concentrated 4000 times on an Atlantic HLB-M Disk using HORIZON SPE-DEX 4790 (USA) with a 47 mm disk holder, in accordance with the extraction programme presented elsewhere .−1, and a linear correlation coefficient (R2) was used to assess the linearity. R2 was proved to be equal to or higher than 0.99 for all targeted substances.The chemical method used in the study was validated in terms of linearity, sensitivity, accuracy, and repeatability ,45. Seve−1 standards in effluent wastewater samples was studied. The extraction recovery was evaluated by looking at the ratio between peak area of spiked samples and that of matrix-matched samples. Extraction recovery was calculated by dividing the peak area of the spiked samples by the peak area of the matrix-matched samples spiked at the end of the sample preparation. As target compounds may be present in actual samples, the difference between readings from wastewater, spiked, and matrix-matched samples was used to determine the analyte concentration. In total, 22 compounds showed recovery of 80–120%, 11 compounds showed recovery of 60–80%, and 7 compounds showed poor recovery of below 60%. The method repeatability was measured by intermediate precision, where the relative standard deviation  at 10.0 and 100.0 ng L−1 was measured and was proved to be below 20% for all targets. The limit of detection (LOD) was found to be in the range 0.02–6.1 ng L−1. For quality assurance purposes, the samples were spiked with a list of internal standards to ensure satisfactory extraction and instrumental analysis. To check for contamination, laboratory procedural blanks and field blanks were used, which underwent chemical analysis together with the wastewater samples. The difference between signals from samples and blanks was adopted for the screening.Regarding accuracy, the recovery of 10.0 and 100.0 ng LLevel of confidence of the identification was expressed in levels from 1 to 5, where 5 denotes the exact mass(es) of interest (lowest confidence). Level 4 entails unequivocal molecular formulas; level 3 indicates tentative candidates; level 2 suggests probable structures with diagnostic evidence; and level 1 implies confirmed structures with reference standards . DetaileTo avoid overlooking antibiotics, all samples were screened for the suspect list of antibiotics and their major TPs  usinSuspected antibiotics were semi-quantified based on the target compound with the highest structural similarity, using the standard addition calibration curve of the target compound in spiked effluent wastewater . For insThe risk assessment was performed on the detected target and suspect antibiotics based on the prioritization methodology established by the NORMAN ,55. The site) of an analyte above the lowest PNEC. EoE represents the ratio between the 95th percentile of all MECsite values per analyte (MEC95) and the PNEC of the analyte, which shows the extent of the effects expected. The risk score was established as the sum of FoA, FoE, and EoE, each scaled from 0 to 1. For prioritization purposes, analytes with a risk score over 1 were prioritized. The remaining substances were considered to pose less risk to the ecosystems. Further details of the prioritization scheme could be found elsewhere [Risk score was established as the sum of three indicators (0–1 for each indicator): (i) Frequency of Appearance (FoA); (ii) Frequency of PNEC Exceedance (FoE); and (iii) Extent of PNEC Exceedance (EoE). FoA considers the frequency of monitoring sites where the analyte was detectable (above LOD). FoE denotes the frequency of sampled sites with maximum observations–maximum environmental concentration (MEClsewhere .Wide-scope screening of 676 antibiotics and its main TPs in the effluent wastewater samples in 11 countries across Europe resulted in the detection of 47 antibiotics, of which 32 were detected by target screening and 15 additional ones were detect by suspect screening. Azithromycin, erythromycin, clarithromycin, ofloxacin, and ciprofloxacin were prioritized as they each entailed risk score of above 1. These ranked compounds are of regulatory interest.Sulfapyridine, trimethoprim, lincomycin, flumequine, metronidazole, sulfamethoxazole, oxolinic acid, and amoxicillin detected in target screening and 8-hydroxyquinoline, clindamycin, linezolid, fluconazole, sulfasalazine, mycophenolic acid, roxithromycin, and cordycepin detected in suspect screening all showed FoA over 0.5, indicating that each of them were detected in over half of the WWTPs investigated. They were widespread in wastewater in European countries. Target screening of these suspected antibiotics would provide a more robust quantification for risk assessment. Wide-scope screening of antibiotics and TPs in a wider range of WWTPs across Europe would reveal more information on the occurrence profile of these chemicals across the continent and “hotspots” of antibiotic contamination, which is essential to develop a regulatory monitoring framework on antibiotics and TPs in Europe. An investigation into the occurrence of antibiotic-resistant genes and effect-based assessment at aquatic ecosystems is recommended to reveal the impacts of antibiotic contamination, especially at “hotspots” that are highly polluted by antibiotics.Total antibiotic load per WWTP spanned from below 1 g/day to over 7000 g/day, which was related to the population that the WWTP serves and its flow rate. Over 1 kg each of azithromycin, clarithromycin, erythromycin, sulfadiazine, and trimethoprim are discharged into freshwater ecosystems every day from the 48 WWTPs investigated in total. The median total load for a given antibiotic across the 48 WWTPs was 35.85 g/day, while the median individual antibiotic load per WWTP was 0.59 g/day. The high antibiotic load reflected the urgent needs for the establishment of regulatory limits on the emission of antibiotics.


Broad-spectrum cephalosporins are rarely recommended as first-line therapy for common pediatric infections, except in some instances of penicillin allergy. Cefdinir in particular is frequently prescribed despite its less optimal pharmacokinetics and pharmacodynamics. The overprescription of broad-spectrum cephalosporins leads to the development of antimicrobial resistance, unnecessary cost, and adverse side effects. Prior studies looking at antibiotic use often solely use electronic medical record (EMR) codes to analyze data; however, limitations exist with this method, and more robust data can be obtained through chart review. In this study, we sought to determine the frequency and indications for broad-spectrum oral cephalosporins prescribed in ambulatory settings at a large, freestanding, children’s hospital network.Antibiotics are prescribed in ∼21% of pediatric ambulatory visits nationally; of those, 16% are broad-spectrum cephalosporins (second–fourth generation). Raw data were generated at the Children’s Hospital Colorado. Derived data supporting findings of the study are available from N.M.P. upon request.We conducted a retrospective observational cohort study by EMR chart review of broad-spectrum oral cephalosporins prescribed in ambulatory settings within a freestanding children’s hospital network from January 1 through December 31, 2019, where >750,000 ambulatory encounters occurred. Encounters from the emergency department, urgent care, general, and subspecialty clinics for patients aged ≤26 years with broad-spectrum oral cephalosporin prescriptions  were included. Encounters from the hematology–oncology clinic were excluded. Data were stored using REDCap.Variables collected included age, antibiotic prescribed, antibiotic indication, allergies, recent antibiotic use (within 30 days), and pertinent microbiological culture results. Descriptive statistical analyses were used to evaluate frequency of prescriptions, antibiotic indications, and variables influencing antibiotic choice. This study was exempt from institutional review board review.In total, 1,381 encounters were included in the study. The median age was 3.9 years . The most common cephalosporin prescribed was cefdinir (81.3%), followed by cefpodoxime (7.1%), cefprozil (6.6%), cefuroxime (2.8%), and cefixime (2.2%). The most common antibiotic indication was otitis media (67.7%), followed by sinusitis (7.2%), pulmonary infections (6.4%), urinary infections (6.2%), ear, nose, throat infections other than otitis media or pharyngitis (5.1%), multiple diagnoses (3.0%), other diagnoses  (2.8%), and pharyngitis (1.5%). Most broad-spectrum cephalosporins were prescribed in the emergency department or urgent-care settings (73.6%). Compared to general care and/or subspeciality clinics, in the emergency department or urgent care, broad-spectrum cephalosporins were prescribed more frequently for acute otitis media (75.7% vs 45.3%) and cefdinir was prescribed less frequently . Of all encounters, 741 (53.7%) had a penicillin allergy documented; of these, 17.9% were listed ≥5 years preceding the encounter. Penicillin allergy descriptions varied, and most were described as a rash (77.9%). Only 0.5% of allergy descriptions reported anaphylaxis. Recent antibiotic use occurred in 438 (31.7%) encounters. For otitis media (n = 934), penicillin allergy and preceding antibiotic use were common indications for broad-spectrum cephalosporin prescriptions  is recommended.Cefdinir was the most common cephalosporin prescribed. Compared to amoxicillin or other cephalosporins, cefdinir is poorly absorbed and has a short half-life, which contributes to inadequate treatment of infections and development of drug-resistant bacterial strains. Allergy descriptions in this study varied and provide an opportunity to further investigate. Rash without other anaphylaxis criteria may be amenable to rechallenge and prevent unnecessary broad-spectrum cephalosporin prescriptions.Perceived penicillin allergy was a predominant reason for broad-spectrum cephalosporin prescriptions, suggesting allergy delabeling as an improvement target. Although ∼10% of US patients report a penicillin allergy, <1% have a true allergic reaction.Limitations inherent to retrospective studies include incomplete data that may skew frequencies ; however, our method of chart review minimizes incomplete information. Other factors difficult to infer from chart review, such as provider preference, pharmacy availability, and insurance coverage, could have contributed to prescription choice. We did not analyze all encounters in which any antibiotic, including broad-spectrum cephalosporins, was prescribed in the study. Finally, antibiotic prescription appropriateness was not assessed .Broad-spectrum cephalosporins are frequently prescribed in ambulatory settings but often not indicated. Our findings highlights penicillin allergy labels and cefdinir prescribing as specific targets to improve antibiotic prescribing. Utilization of penicillin delabeling pathways in ambulatory settings, provider education about pharmacokinetics and pharmacodynamics of oral cephalosporins, and point of care electronic prescribing modifications  should be next steps for improved prescribing practices.