Echinococcus multilocularis) that causes alveolar echinococcosis, domestic and wild carnivores act as definitive hosts, and rodents act as intermediate hosts. The presented study contributes to the research on the distribution and transmission pattern of E. multilocularis in China having identified sheep as an unusual intermediate host taking part in the domestic transmission of alveolar echinococcosis in Gansu Province, China.In the normal life cycle of the parasite (mt) genome and selected nuclear genes were amplified from the cyst-like lesion for identification. To confirm the status of the specimen, comparative analysis with reference sequences, phylogenetic analysis, and network analysis were performed.From 2020 to 2021, nine whitish different cyst-like were collected from the liver of sheep in Gansu Province for examination. A near complete mitochondrial (E. multilocularis NADH dehydrogenase sub-unit 1 (nad1) (894 bp) reference sequences deposited in GenBank. Furthermore, amplification of the nad4 and nad2 genes also confirmed all nine samples as E. multilocularis with > 99.30% similarity. Additionally, three nuclear genes, pepck (1545 bp), elp-exons VII and VIII (566 bp), and elp-exon IX (256 bp), were successfully amplified and sequenced for one of the isolates with 98.42% similarity, confirming the isolates were correctly identified as E. multilocularis. Network analysis also correctly placed the isolates with other E. multilocularis.The isolates displayed ≥ 98.87% similarity to E. multilocularis in an unusual intermediate host, which is considered to have the highest zoonotic potential, the result clearly demonstrated the necessity for expanded surveillance in the area.As a result of the discovery of The online version contains supplementary material available at 10.1186/s40249-023-01120-0. Echinococcus multilocularis, rodents and other small mammals  serve as intermediate hosts, while foxes such as red foxes (Vulpes vulpes), Arctic foxes (Alopex lagopus), Tibetan foxes (V. ferrilata) and sand foxes (V. corsac) serve as definitive hosts [E. multilocularis metacestodes produce a multilocular (multichambered) mass that contains hundreds to thousands of protoscolices in some and fewer to none in others [In the well-known wildlife transmission cycle of ve hosts , 2. In cve hosts . In the n others .E. multilocularis is not uniform, with areas with high and low prevalence zones. These disparities have been linked to the use and structure of the landscape , as well as climatic elements (temperature and humidity), which determine the species range and abundance of rodent intermediate hosts [E. multilocularis is expanding both its geographic and host range as a result of anthropogenic influences and has been linked to changes in the distribution of definitive hosts [E. multilocularis tapeworms can be found in 1–60% of wild canids (particularly foxes) and less than 1% of dogs and cats. In China, up to 12% of dogs can be infected with E. multilocularis, but the cysts are commonly found in less than 0.5% to 10% of livestock [In many countries, the distribution of te hosts . More sove hosts –8. Depenivestock .E. granulosus sensu lato and E. multilocularis have multi-host systems that are influenced by environmental factors such as the number of host species, habitats, density, predator–prey relationships, and egg survival [Generally, both survival –16.Alveolar echinococcosis (AE) is common in the northern hemisphere, including western, northern, and eastern Europe, Russia, western Alaska, the northwestern portion of Canada, central Asia, northern Japan, and western and northern China –21. In CmtDNA) markers, the microsatellite EmsB, and a multi-locus nuclear DNA markers were applied to E. multilocularis genotyping studies [E. multilocularis is less diverse than E. granulosus sensu lato, recent taxonomic studies have shown intra-specific variations within E. multilocularis at the global scale, allowing the identification of four genetic subgroups that are restricted to specific geographic regions: European, North American, Asian, and Mongolian [Genetic characterization using mitochondrial deoxyribonucleic acid  mitochondrial (mt) gene (~ 1280 bp) using forward (5′-ATTATAGAAAATTTTCGTTTTACACGC-3′) and reverse (5′-ATTCACAATTTACTATATCAAAGTAACC-3′) primers [https://blast.ncbi.nlm.nih.gov/Blast.cgi) for identification. Thereafter, a near-complete mt genome (Table During a routine cystic echinococcosis (CE) investigation suspected (whitish lesion) samples of nine sheep from different slaughterhouses were collected. The infected livers were separated from the carcass. Then the cyst-like samples were separated from the infected liver and washed three times with phosphate buffered saline; thereafter, they were kept at – 20 ℃ until DNA extraction. Each sample was treated as a separate isolate. Following the manufacturer's instructions, total genomic DNA was extracted using the TIANamp Genomic DNA Kit . PCR amplification and sequencing were achieved for the NADH dehydrogenase subunit 1 ( primers . This wa2+ plus) and 2 µl of deoxynucleoside triphosphate (dNTP) mixture (2.5 mmol/L each) in a final concentration of 100 µmol/L, 0.5 µl Prime STAR GXL DNA Polymerase (0.625 U/25 µl) , 0.5 µl (10 pmol) of each primer with a final concentration of 0.2 μmol/L each, 0.5 μl of 20–50 ng of purified genomic DNA, and nuclease-free water up to the final volume of 25 µl.Each primer pair was used in a separate polymerase chain reaction (PCR) reaction. The reaction contained 5 µl 5 × prime STAR GXL Buffer , and 72 ℃ for 2 min; then 25 cycles of 95 ℃ for 30 s, 55 ℃ for 30 s, and 72 ℃ for 2 min; and a final elongation step at 72 ℃ for 10 min modified after Laurimäe et al.  was usedGel electrophoresis (1.5%) made from 1 × TAE  stained with TS-GelRed  was used to visualize 5 μl of the amplified PCR products. The amplicons were sequenced afterwards  using the same set of primers.Nucleotides were inspected for errors in UGENE program . The spenad1, cob, nad2, nad5, and the near-complete mitochondrial genome datasets. Taenia solium (accession number: AB086256 and NC_004022) was used as the outgroup. Computation of the population diversity and neutrality indices, such as Tajima's D [Taenia solium (NC_004022) was used as an outgroup for the estimation of Fu’s indices. For the sequence data of the cob and nad2 genes, each of which has more than five haplotypes, diversity and neutrality indices were calculated. Finally, PopART was used to visualize the haplotype and networks [Phylogenetic analysis was repeated for jima's D  and Fu'sjima's D , was achjima's D  and expojima's D . Taenia networks .Echinococcus cysts. They were all located in the liver, small in size, and without cystic fluid  and cytochrome c oxidase subunit 1 (cox1) genes. Only one specimen was amplified using cox2, nonetheless sequencing was unsuccessful due to poor DNA quality.As shown in Table nad1 (894 bp), cytochrome b (cob) gene (1068 bp), nad2 gene (874 bp), and nad5 gene (1575 bp) sequences' BLAST results showed 98–100% similarity to the E. multilocularis reference sequence . Similarly, the concatenation of nine mt genes was > 99.3% similar to the E. multilocularis reference sequence . Likewise, all three nuclear genes; pepck (1545 bp), elp-exons VII and VIII (566 bp), and elp-exon IX (256 bp), were only successfully sequenced for a specimen and confirmed the infection was actually caused by E. multilocularis with 98.42–99.93% similarity to reference sequences . All resulting nucleotide sequences have been deposited in GenBank under the following accession numbers: OR047885–OR047893 (nad1), OR047894–OR047899 (cob), OR047900–OR047905 (nad5), and OR047906–OR047914 (nad4).The nad1 (894 bp) sequences confirmed the species status of all the isolates as E. multilocularis, as they formed a clade with E. multilocularis from other hosts and locations , cob (1068 bp), nad5 (1575 bp), and 7005 bp concatenated sequences of 8 genes  , cob 108 bp, nadnad1 (894 bp) sequences showed two singleton variable sites and two parsimony informative sites  showed more mutation sites (27 singletons and 93 parsimony-informative sites). Haplotype Hap_7 was common  in the population and was mainly composed of European (n = 10) isolates. A variety of haplotypes appeared as a radial network through various mutational steps , haplotypes (Hap_5 and Hap_12) were common, each making up 19.2% (10/52) of the population  showed eight mutations (two singletons and six parsimony-informative sites), and some of the present isolates share the same haplotype with previously described Chinese and Japanese isolates. Finally, the concatenated sequences of 7005 bp  showed 120 mutations (41 singletons and 79 parsimony-informative sites) . The haplotype diversity index for the population (n = 47) was 0.932, the nucleotide diversity index was 0.01267 while Tajima’s D (− 1.89298) had a statistically significant P-value (P < 0.05), and Fu’s Fs (− 0.98675) were not statistically significant (P > 0.10) when T. solium (NC_004022) was used as an outgroup. Similarly, there were also 18 distinct haplotypes of the nad2 gene (874 bp). The haplotype diversity index for the population (n = 52) was 0.896, the nucleotide diversity index was 0.00633, Tajima’s D (− 1.76799) had a statistically significant P-value (P < 0.05), and Fu’s Fs (− 2.78572) were not statistically significant (P > 0.10) when T. solium (NC_004022) was used as an outgroup.There were 26 distinct haplotypes of the Echinococcus species E. granulosus sensu stricto (E. granulosus s.s.), E. multilocularis, and E. shiquicus coexist [E. granulosus s.s. was confirmed to be mainly transmitting among synanthropic hosts, such as dogs and livestock, while transmission patterns of E. multilocularis and E. shiquicus entail intricate sylvatic cycles involving a variety of animal species [In some parts of China, the  coexist –42. Mean species .E. multilocularis metacestodes have been found in natural settings in a range of mammalian hosts that are not involved in the transmission cycle, such as horses, domestic and wild pigs, nutria (Myocastor coypus), various species of captive monkeys, and others [E. multilocularis lesions [Previous findings have indicated that d others , 45. A s lesions . The nec lesions . Similar lesions , as stat lesions . Molecul lesions . In the  lesions . Additio lesions . In the  lesions .E. multilocularis. Inline to this, Zhao et al. [E. multilocularis infection in South Gansu Province, China [E. multilocularis in domestic animals such as sheep in endemic areas.In the current finding, based on the near-complete mitochondrial and nuclear gene analysis, sheep were found to be infected by o et al.  have repe, China , suggeste, China . These fEchinococcus cysts, suggesting that aerosol transmission, which causes lung cysts, is a potential possibility [E. multilocularis is endemic [E. multilocularis in the present study.On the other hand, previous reports showed that experimentally introduced eggs in lambs' lungs had the ability to hatch and develop into sibility , 54. The endemic . This coE. multilocularis isolates from several intermediate and final Asian hosts form a well-defined, geographically characteristic clade that is distinctly different from isolates from Europe and North America [Echinococcus species are getting established in western China [E. canadensis and E. multilocularis have only recently been detected in wolves.Inferred from molecular and phylogenetic data,  America . As was rn China –42, so irn China  from CanThe current study's unfortunate limitations are the lack of histopathology of the lesion structure and the maintenance of the samples for further processing. The study also failed to provide a clear explanation of how the transmission might have occurred.Echinococcus cysts. The cysts, which were discovered in the livers of sheep, were small white patches devoid of cystic fluid. The present isolates revealed that the infection in sheep was caused by E. multilocularis based on near-complete mitochondrial and nuclear gene analysis, but we were unable to provide a clear explanation for how the transmission could have occurred. In endemic areas where CE and AE are prevalent, it is recommended to take into account that domestic animals, such as sheep, could be infected by E. multilocularis. As a result, given the importance of the parasite to public health, there is a need for increased surveillance in the region in particular and in the country in general following the detection of E. multilocularis in an uncommon intermediate host, which is known to have the highest zoonotic potential.The cysts did not have the same morphological characteristics as conventional Additional file 1:Figure S1. Geographical administrative division at the municipality level, Gansu Province, China.


However, the relationship between air pollution and these cancers in regions of low pollution is unclear. We investigated associations between fine particulate matter  were followed from 1996–1999 until 2018. Pollutant concentrations, as a time-varying variable, were estimated at participants’ residential addresses using land use regression models. Incident lung and bladder cancer were identified through the Western Australian Cancer Registry. Risks were estimated using Cox proportional-hazard models , adjusting for smoking, socioeconomic status, and co-pollutants.2.5 and BC in the adjusted single-pollutant models. A weak positive association was observed between ambient air pollution and squamous cell lung carcinoma but not lung adenocarcinoma. Positive associations were observed with bladder cancer, although these were not statistically significant. Associations were attenuated in two-pollutant models.Lung cancer was associated with PMLow-level ambient air pollution is associated with lung, and possibly bladder, cancer among older men, suggesting there is no known safe level for air pollution as a carcinogen. Lung and bladder cancers are significant causes of mortality and morbidity in Australia and worldwide –3. AmbieLung cancer is the fifth highest cancer diagnosis and a leading cause of cancer-attributable mortality in Australia . Whilst 2) and lung cancer have been identified in regions in Europe . Th. Th47]. Two histological subtypes of lung cancer were investigated, categorised by ICD-O-3 histology codes. These were: SCC (8051–8084) and adenocarcinoma . These subtypes accounted for 50% of all lung cancers observed. Other histological groups of lung cancer  were not studied separately, as there were too few events.http://www.dagitty.net/ . These are described in Supplementary Figs. Data were collected at recruitment using questionnaires and health assessments, and the following baseline data were considered for analyses: age, height and weight, socioeconomic status (SES) and the self-reported variables: country of birth, smoking status, alcohol consumption, physical activity . The SESn = 43) and SES (n = 5), leaving 11,679 study participants. For the lung cancer analyses, we excluded participants with lung cancer diagnosed prior to HIMS recruitment (n = 62), leaving 11,617 participants for this analysis. Similarly, for bladder cancer, participants with previous diagnoses (n = 52) were excluded, leaving 11,627 participants for the bladder cancer analysis.Of the 12,203 participants in the original HIMS, because our air pollution estimates were restricted to the Perth Metropolitan Area, 476 men were excluded for this reason , we also adjusted for each of the other two pollutants, in separate models. All two-pollutant models were also adjusted for baseline SES and smoking status.Two-pollutant models were developed for PM2.5 concentrations of 3, 5, and 7 µg/m3 are compared with a reference value of 1 µg/m3, NO2 concentrations of 10, 15, and 25 µg/m3 are compared with a reference value of 5 µg/m3, and BC concentrations of 0.8 × 10−5, 1.2 × 10−5, and 1.6 × 10−5 m−1 are compared with a reference value of 0.5 × 10−5 m−1.The proportional hazards assumption was assessed using plots of Schoenfeld residuals, and no violations were detected. To allow for non-linearity in the relationship between each pollutant and each cancer outcome, we considered models where each pollutant was entered as a restricted cubic spline with three knots. Knot locations were determined by the recommendations in Harrell . When moFive sets of sensitivity analyses were conducted. The first was assigning a baseline pollutant exposure to all participants. This was the average concentration of each pollutant during the participant’s recruitment year. Current studies have used baseline concentrations to estimate participant exposures to ambient air pollutants , 38, 43.When using age as the timescale, comparisons are made between participants at specific ages, as opposed to specific calendar years of exposure. As such, we conducted a sensitivity analysis which included an interaction between birth cohort (birth years 1914–1924 and 1925–1932) and each air pollutant to see whether the impact of air pollution on lung and bladder cancer differed for those born at different times.The third set of sensitivity analyses excluded participants who had a lung or bladder cancer diagnosis in the first two years of follow-up, in addition to those who were excluded due to diagnoses prior to recruitment. This factored in lung or bladder cancer cases at baseline that may have had delayed diagnosis or registration to the WA Cancer Registry.Exposure estimates were based on residential addresses at the time of recruitment, thus assuming that all participants had lived at the same residential address the whole time, and as such, a fourth set of analyses was done to exclude participants who had moved addresses (‘movers’) during the follow-up period. Participants who had moved addresses from the baseline during the study were recorded in the HIMS dataset.Finally, we also included all available covariates from the DAGs in the primary analyses to assess the robustness of our models.All analyses were performed using Stata software package 17.0 .Baseline demographic and lifestyle characteristics of the study participants are presented in Table 3 for PM2.5, 15.83 µg/m3 for NO2, and 1.06 × 10−5 m−1 for BC. Average concentrations at the end of the study (2018) were: 4.35 µg/m3 for PM2.5, 11.20 µg/m3 for NO2, and 0.81 × 10−5 m−1 for BC. Further distributional information is described in Supplementary Table r) <0.7  were: 5.65 µg/mThere were 692 (6.0%) lung cancer events identified during the study period, with a total follow-up time of 158,479 person-years.2.5 and BC, but not NO2, were mostly statistically significant. In the two-pollutant models, the results for PM2.5 were attenuated, and lost statistical significance, when controlling, separately, for NO2 and BC. The results for BC were attenuated when controlling for PM2.5 but not for NO2, but stayed statistically significant.Positive associations between the pollutants and lung cancer were observed in both crude and adjusted models Table . In the 2.5 and BC of participants were positively associated with both SCC and adenocarcinoma, across the unadjusted and adjusted single-pollutant models, although these were not significant. The HRs in the respective two-pollutant models were further attenuated, except for BC when controlled for NO2. Concentrations of NO2 were positively associated with SCC, but not ADC in the single-pollutant models. No associations between incident lung cancer and NO2 were observed across all two-pollutant models.In the lung cancer cohort, there were 138 (1.2%) SCCs and 201 (1.7%) adenocarcinomas of the lung reported during follow-up Table . Concent2.5 and BC were positively associated with bladder cancer in their respective single and two-pollutant models, although these were not statistically significant , during 158,440 person-years of follow-up. Concentrations of PMnt Table . A weak We observed evidence of a non-linear relationship between pollutants and both lung and bladder cancer Fig. .Fig. 1GrWhen stratified by smoking status, positive associations were observed between all pollutants and lung cancer for the former smokers’ group who had quit smoking ≥10 years ago in the single-pollutant models, but not consistently in other smoking groups .For both lung and bladder cancer analyses, no significant interactions were observed between ambient air pollutants and the birth cohorts , respectively [We observed positive associations between follow-up exposures to PMectively ), and loectively , 35, 36.2.5 was 4.63 µg/m3 (0.01–9.20 µg/m3), NO2 was 13.59 µg/m3 (0.82–29.02 µg/m3), and BC was 0.98 × 0.5 × 10−5 m−1 (0.08–2.03 × 10−5 m−1). Our findings support the notion that there may not be a threshold concentration for the carcinogenic effects of ambient air pollution.The range of exposure concentrations for each pollutant reported in our study was narrow. The annual average concentrations (and range) during follow-up for: PM2.5 and lung cancer were consistent with recent studies conducted in the USA [2.5 exposures. For example, the ELAPSE study [3, and a pooled-HR of 1.13 (95% CI: 1.05–1.23) per 5 µg/m3 increase in PM2.5. Studies in Canada with lower exposure concentrations reported HRs of 1.02 (95% CI: 1.01–1.05) per 5.3 µg/m3 [3 [3 [2.5 concentrations were 7.68 µg/m3 (mean), 7.4 µg/m3 (mean), and 9.1 µg/m3 (median). On the other hand, the Biobank study [3 increase in PM2.5 with mean PM2.5 concentration of 10 µg/m3. Recent studies (with a wider exposure range) have suggested that the relative risk of lung cancer  may be greater at lower concentrations of PM2.5, compared to higher concentrations [The associations between PM the USA , 35, Can the USA –19, and  the USA , 50, wheSE study , a pooleµg/m3 [3 , and 1.3m3 [3 [3 , where atrations , 17, 19.2 and lung cancer has regularly been observed [2, significant associations with lung cancer have been found in studies conducted in the Netherlands [2 reported in these studies were below European Air Quality Standards of <40 µg/m3 [2 concentrations were below the Australian standard of 28 µg/m3. We observed non-significant associations between NO2 and lung cancer, which was a similar finding to a pooled analysis of European cohorts in relatively low-pollution areas  [The association between ambient NOobserved , 50, 51,observed , 28. In herlands –25, the herlands , and Canherlands , 19. The40 µg/m3 . In our 97–1.07) .We observed significant associations between BC and lung cancer in all models. There is currently a paucity of studies on BC and lung cancer. We identified three studies and one pooled analysis that investigated associations between BC and lung cancer , 25, 33.In 2012, the IARC declared diesel engine exhaust as a Group 1 human carcinogen . Ambient2 and BC and lung cancer, when analysed together, a positive, but non-significant association, with lung cancer remained with BC but not with NO2. This suggests that the association of NO2 with lung cancer is in part reflecting the effects of BC. The HRs were stronger when NO2 was included in the model with BC, suggesting there may be additive effects. As both NO2 and BC are produced from traffic-related pollution, they are often present together [Whilst positive associations were observed between both NOtogether .2.5 and both histological lung cancer subtypes in the single-pollutant models. The effects were attenuated in the two-pollutant models, particularly for lung adenocarcinoma. Concentrations of NO2 were positively associated with increasing risk of SCC; however, effects were also attenuated after controlling for other pollutants. No associations were observed for NO2 with adenocarcinoma.The relationship between histological subtypes of lung cancer and ambient air pollution remains unclear. Positive associations were observed between BC and both SCC and adenocarcinoma, across both single and two-pollutant models. Positive, but non-significant, associations were also observed for PM2.5 and adenocarcinoma have been observed, compared with other lung cancer subtypes [2.5 and adenocarcinoma, but not with SCC [2 or BC, with either adenocarcinoma and SCC [2 or black smoke. These studies used single-pollutant models and did not adjust for co-pollutants. The risk of incident SCC (and not adenocarcinoma) was not found to be associated with PM2.5, NO2 and BC in previous studies.Our findings contrast the results from previous studies, where stronger associations between PMsubtypes , 43. Thewith SCC . However and SCC . Hart et and SCC . also re2.5 and bladder cancer, although this was not statistically significant. This is consistent with the multi-centre European study, the ELAPSE Study [There was a positive association between PMSE Study , where aSE Study  and in aSE Study . HoweverSE Study , the USASE Study , and anoSE Study .2 were not associated with bladder cancer, which is consistent with existing literature. No associations with bladder cancer were reported in studies in Spain [2 and BC were not independent of each other, and the presence of both in the co-pollutant analysis resulted in attenuation of their respective effects on bladder cancer.Exposures to NOin Spain , the USAin Spain , and a min Spain . Howeverin Spain , where n2.5, NO2 and BC and both respective cancers remained consistent in the sensitivity analyses  compared to the full cohort  [We observed modest effect modification by smoking status in the lung cancer analysis, and this has also been observed in the literature , 22, 35.-smokers , 15. Anomprecise . AnotherSmoking status did not modify the effect of ambient air pollutants and bladder cancer in our study. Our findings were similar to other studies, where a non-significant interaction between smoking status  and packOur study had several strengths. The follow-up time of 22 years was suitable for lung and bladder cancer, which both have long latency periods. Analysing data from an older-aged cohort is a consideration of a population particularly vulnerable to the health effects of hazardous air pollution. The HIMS is linked to the WADLS and follow-up data for all participants were available through data linkage, including to state hospital records and mortality registry. Reporting cancer diagnoses to the WA Cancer Registry is also a mandatory requirement for WA health practitioners . The WAD2.5, NO2 and BC concentrations in Perth by approximately 67%, 69% and 85%, respectively [The validated LUR models had good performance, and could explain variability in PMectively , 42. HowThe HIMS cohort only included older, mainly Caucasian men, limiting generalisability. Many studies in the literature have used large-scale cohorts , anAlthough we reported a large number of lung cancer events, the case counts for bladder cancer and histological subtypes of lung cancer were relatively fewer. This could limit the statistical power of these analyses. Whilst our sample size was relatively small compared to other studies, our cohort was a subset of the population most vulnerable to lung (and bladder) cancers. Evaluating lesser-known cancer risk factors such as air pollution was, therefore, suitable and relevant for our cohort.Pollution estimates were from residential addresses and did not consider where they spent their time. Personal exposure to air pollution is dependent on the day-to-day microenvironments of the individual, which include both indoor and outdoor exposures at their home .Other residual confounding for lung cancer risk may not have been considered. For example, the UK Biobank Study observed that genetic factors could increase the effect of ambient air pollution on lung cancer by 50% [In conclusion, we found an increased risk of lung and possible increase of bladder cancer in a cohort of older men in Perth, WA, residing in areas where ambient air pollution concentrations are typically considered very low. This adds to the increasing evidence that negative health effects are being identified at low concentrations of air pollution. Further studies are needed to understand the carcinogenic potential of ambient air pollution at low concentrations.Supplementary Information


Basal values of hematological and serum biochemical parameters are necessary for health and animal production performance evaluation. Therefore, considering the importance of cattle production in the Brazilian savannah area and the lack of regional data, we aimed to know the differences related to age, breed group, the influence of the productive system, and geographic region. We established baseline values for beef calves at weaning age. Jugular blood samples were collected in a single instant from 30 calves from each of the following breeds: Nellore, Senepol, Nellore × Aberdeen Angus cross, Nellore × Senepol cross, and Nellore × Aberdeen Angus × Senepol cross. Hematological and serum biochemical evaluations and a proteinogram were performed according to routine techniques. In conclusion, the available reference intervals for healthy animals can be used routinely without interference from the geographic region for animals produced without nutritional failures since changes are registered in pathological, infectious, metabolic, or nutritional deficiency situations. Furthermore, these results will help to understand the adaptation advantages and the possible physiological processes of those breeds and their crosses in the savannah biome of Brazil.The Brazilian savannah region, characterized by high average temperatures, well-defined rainy and dry seasons, soil with low productive potential, and high pressure for parasitic diseases, is home to the highest percentage of the beef herd, which is the world’s largest commercial beef producer. Therefore, breeds that present rusticity combined with productivity are the focus of research in cattle breeding in the region. Considering their geographic particularities and their effects on the animals’ blood parameters, the objective was to study the behavior of hematological variables, serum biochemistry, and acute phase proteins in beef calves at weaning age. Jugular blood samples were collected in a single day from 30 weaning calves (about eight months old and 200 kg of body weight) from the following breeds: Nellore, Senepol, Nellore × Aberdeen Angus cross, Nellore × Senepol cross, and Nellore × Aberdeen Angus × Senepol cross. Hematological data were obtained using an automatic cell counter, serum biochemical measurements were obtained using commercial kits, and the electrophoretogram was obtained using the SDS-page technique. In general, the results were consistent with data already published in similar situations regarding health status, age, and level of metabolic activity. However, differences observed between groups can be explained by differences observed in other concurrent variables like temperament. The pure zebu breed was more reactive than pure taurine. While crossbreds showed intermediate values, and parasitic infestation, the pure taurine breed with higher parasite infestation, while the zebu breed had lower values, which produced effects in some laboratory tests, and generated differences between breeds. In conclusion, the reference intervals available for healthy animals can be routinely used without interference from the geographic region for animals produced without nutritional failures as long as changes are recorded in pathological, infectious, metabolic, or nutritional deficiency situations. However, it is suggested that a study covering a larger number of herds may demonstrate a greater geographic effect on the studied variables. Brazil is the largest exporter of beef in the world , and itsThe state of Tocantins belongs to the northern region of Brazil and is part of the Tocantins–Araguaia Basin. Its main economic activities are livestock, forestry, and grain production, mainly soy, corn, and rice, with considerable potential for expansion since about 50% of the soil is suitable for agriculture . CurrentLaboratory diagnosis consists of comparing the patient’s serum hematological and biochemical values with reference ranges obtained from clinically healthy animals ,8, thereThe acute phase reaction occurs in response to infection or tissue damage induced by endogenous or exogenous factors—damage-associated molecular patterns (DAMPs) . It is cAdaptive responses to environmental temperature affect productive performance, as nutrients that would be useful for body development and milk production will be diverted to thermoregulatory mechanisms, causing a reduction in dry matter intake, consequently lower rates of daily weight gain and greater susceptibility to infectious and parasitic diseases . Calves Therefore, this study aimed to determine the mean values for the hematimetric and clinical biochemical variables in calves at the weaning age of the main beef breeds raised in the savannah biome in Brazil.The primary objective was to determine the baseline values for hematological, biochemical, and metabolic variables and the expression of acute phase proteins (APPs) in healthy weaned beef calves of the main races produced in the state of Tocantins, correlate these data with the degree of endoparasite infestation, the level of reaction to external stimuli, and the concordance of the results with those already demonstrated in the literature.The experimental units consisted of healthy cattle between six and eight months old, in the rearing stage, produced in a single rural establishment located in the municipality of Monte Santo do Tocantins, state of Tocantins, Fazenda Chão Mineiro. The property has 1674.7 ha, an altitude of 300 m above sea level, geographical coordinates of 9°59′56″ S and 49°06′40″ W, average annual rainfall of 2038 mm, and an average temperature of 26.8 °C .Panicum spp. and Brachiaria spp. in groups whose size was determined by the size of the paddock. Water and commercial 80P (8% of phosphorus) mineral supplement ad libitum and grain concentrate (DM (dry matter): 89.68%, TDN : 65.18%, CP (crude protein): 25%, EE : 2.44%, Ca : 1.22%, P (phosphorus percentage): 1.09%), about 1.5 kg/animal/day (0.3% BW/day (bodyweight/day)) daily in the morning, receiving immunization against foot-and-mouth disease, following the sanitary calendar in force, clostridia, and respiratory diseases, as well as endectoparasiticide control with macrocyclic lactones and benzimidazole. Each experimental group corresponded to a breed type: NE (Nellore breed) (n = 31), SE (Senepol breed) (n = 30), NE×AN (double-cross Nellore/Aberdeen Angus) (n = 34), NE×SE (double-cross Nellore/Senepol) (n = 34), and Tricross  (n = 31), and therefore a total of 160 subjects, and each group had at least 30 uncastrated male individuals subjected to a blood draw at a date close to weaning.Animals were kept on extensively cultivated pastures of The animals’ health was confirmed by individual inspection in the field, considering that the systemic response to stressful stimuli, such as restraint, is individual and may impair the clinical evaluation . On inspTaking into account the diversity of the breed groups that were involved in the study and the possibility of correlation with the hematological and serum biochemical data, a behavioral evaluation was performed to establish a temperament score, from which the methodology was adapted . The adaAs for ReS, it was measured at the moment of the animal’s release from the cattle crush and consisted of grades 1, 2, 3, 4, and 5 to curb the concentration of intermediate grades. This score is stratified in g for 10 min [Peripheral blood samples were obtained by jugular venipuncture before de-feeding using the BD Vacutainer vacuum system  and 40 × 0.9 mm needles preceded by local antisepsis by the same vet every time. Blood aliquots were collected in tubes containing dipotassium ethylenediaminetetraacetic acid , 4 mL, for blood count, and in a 10 mL tube with clot activator for obtaining serum, its biochemical analysis, and protein fractionation . Immediar 10 min  and propKnowing the influence of gastroenteric parasite load on haematological parameters, serum biochemistry, and inflammatory responses, the animals were submitted to coproparasitological evaluation using stool samples collected directly from the rectal ampulla of each experimental unit, respecting a minimum of 90 days after the last deworming, because this is when macrocyclic lactones and benzimidazoles, in synergistic use, lose their endectocide effect . The sam6/µL), free hemoglobin concentration , hematocrit , mean corpuscular volume , and ; leukocyte variables: total leukocyte count , relative , and absolute leucocyte formulae ; and platelet variables , all obtained using an ABX VET automated cell counter ABX VET Horiba .The blood count included the erythrocyte variables: erythrocyte count  was manual by analysis of blood smear, stained with a mixture of acid and basic anilines, dissolved in methanol , and reaThe metabolic parameters analyzed, the material, and the methodology used are described in Indirect bilirubin results (mg/dL) were obtained by subtracting the direct bilirubin value from the total bilirubin value. Sodium, potassium, and inorganic calcium values, expressed in mmol/L, were obtained by Roche 9180 Electrolyte Analyser , while the other variables had their results measured with a semi-automatic spectrophotometer LabQuest model , and with the respective commercial kits of the same brand.The following serum protein fractions were quantified: total protein, ceruloplasmin, haptoglobin, albumin, α1GA, and transferrin, by sodium dodecyl sulfate acrylamide gel electrophoresis (SDS-PAGE) . FollowiThe bovine coproparasitological examination was performed according to the McMaster technique , with fep-value < 0.05. When the data did not meet the requirements of ANOVA, even after data transformation, the non-parametric methodology was used, including the Kruskal–Wallis test with a p-value adjusted by the Benjamini–Hochberg method at 5%. The analysis of possible relationships between variables was done with Pearson’s correlation test. The multivariate structure of the data contained in the dataset was explored by dimension reduction  using the function prcomp from R package stats [All statistical analyses were performed with R software . After confirming the homoscedasticity of data by the Levene test and the normality of data by the Cramér Von Mises test, analysis of variance (ANOVA) of quantitative data was done to evaluate the responses. When necessary, a comparison of means of pairs was made using Tukey’s test, with a ge stats ,31,32, gge stats . Additioge stats  implemenge stats ,35.6/µL in the Nellore (NE) group and 8.72 × 106/µL in the Senepol (SE) group (p = 1.269 × 10−4). The hemoglobin (Hb) concentration was between 11.34 g/dL in the Nellore × Aberdeen Angus cross (NE×AN) group and 10.52 g/dL in the SE group (p = 0.0398). The corpuscular hemoglobin concentration (CHC) reached values between 10.68 g/dL in the NE group and 12.37 g/dL in the triple-cross Nellore × Aberdeen Angus × Senepol . For mean CHC (MCHC), the values ranged from 33.51% in the NE×AN group to 34.43% in the SE group; there was no difference between the NE, NE×AN, and Tricross groups, but we found a difference between SE and Nellore × Senepol cross (NE×SE).The total red blood cell (RBC) count ranged between 10 × 10There was no statistical difference between the groups for hematocrit (Ht) values, which ranged between 33.85 and 30.58%; mean corpuscular volume (MCV) ranged between 31.87 and 36.68 fL, and total platelet count (TPC) ranged between 263.81 × 103/µL and 330.81 × 103/µL. p = 3.33 × 10−4), with a significant difference between these and records of different intermediate values in the NE×AN, NE×SE, and Tricross groups. For the release score (ReS), the values ranged from 1.4 (SE group) to 2.39 (NE group) (p = 1.705 × 10−4), with no difference between the SE, NE×AN, NE×SE, and Tricross groups. Then, the sum of these indexes maintained the same behavior, varying between 3.47 in the SE group and 5.77 in the NE group (p = 1.68 × 10−5), showing a significant difference, while the other groups presented intermediate values different from the extremes. All the average values by temperament scores are available in The restraint score (RTS) ranged from 2.07 in the SE group to 3.39 in the NE group (p < 0.05); there was no difference between the NE, NE×AN, NE×SE, and Tricross groups. Birth weight (BrW) showed values between 28.26 (NE×AN) and 31.46 kg (SE group) (p < 0.05), whereas the NE, SE, NE×SE, and Tricross groups did not differ. Body weight (BdW) ranged between 181.68 (SE group) and 225.84 kg (Tricross) (p < 0.05), while NE and NE×AN, NE×SE, and Tricross did not differ. Mean daily weight gain (MDWG), on the other hand, showed values between 0.71 (NE group) and 0.85 kg/day (Tricross) (p < 0.05), and the NE and NE×AN, SE, NE×SE, and Tricross groups showed no significant difference between them, and the medium values are exposed in Age ranged from 183 (SE) to 242 days (NE) (p = 1.676 × 10−5), with only the SE group showing statistical difference compared to the others. Total egg count showed the same behavior as trichostrongylid, varying between 52.94 (NE×SE) and 556.66 epg .The counts of parasite eggs and oocysts in the feces showed a little discrepancy between the groups and are described in 3/µL, NE×AN, to 19.93 × 103/µL, SE (p = 8.7694 × 10−6), and there was no statistical difference between NE, SE, and Tricross, nor between NE×AN and NE×SE. The total neutrophil count (Neu) ranged between 3.3 × 103/µL in the NE×AN group and 5.89 × 103/µL in the SE group (p = 5.7044 × 10−5), while the pairs SE and NE×SE, and NE and Tricross did not differ. Total lymphocyte (Lymph) count showed values between 10.41 × 103/µL in the NE×SE group and 13.13 × 103/µL in the Tricross group (p = 1.7263 × 10−5); there was no significant difference between the NE, SE, and Tricross groups, and between NE×AN and NE×SE. The values observed for total eosinophil count (Eos) ranged between 0.041 × 103/µL, NE×AN and 0.252 × 103/µL, SE (p = 0.291), without statistical difference between the NE×AN, NE×SE, and Tricross groups, while the NE and SE groups differed from each other. The total basophil (Bas) count showed results between 0.011 × 103/µL, SE and 0.037 × 103/µL, NE×SE (p = 2.2616 × 10−6), with only the NE×SE group differing from the others. Total monocyte (Mon) count, on the other hand, showed mean values between 0.54 × 103/µL, NE and NE×SE groups, and 1.01 × 103/µL, SE group (p = 4.6739 × 10−8), and the NE, NE×AN, and NE×SE groups did not differ from each other, and Tricross and SE differed significantly from all others groups.Results for leucometric variables are in p = 1.5077 × 10−4), with the NE, SE, NE×SE, and Tricross groups not differing from each other. For alkaline phosphatase (AP), values ranged between 431.43 UI/L (SE) and 926.58 UI/L (NE) (p = 1.866 × 10−12), while SE, NE×AN, NE×SE, and Tricross groups showed no statistically significant difference. The average values of enzyme concentration are in Serum activity of the enzymes alanine aminotransferase (ALT), creatinine kinase (CK), and γ-glutamyl transferase (GGT) did not differ significantly between groups, showing values between 17.83 UI/L (SE) and 29.61 UI/L for NE, 244.27 UI/L (SE), 641.55 UI/L (NE×SE), 17.35 UI/L (NE), and 20.80 UI/L for SE. Mean aspartate aminotransferase (AST) results were between 54.32 UI/L (NE×AN) and 69.45 UI/L (Tricross) (p = 1.56358 × 10−10), while SE and NE×AN did not differ and NE×SE differed from all others showing intermediate value. For urea concentration, there was variation between 15.94 mg/dL (NE×AN) and 21.73 mg/dL (SE) (p = 6.3264 × 10−5), while NE, NE×AN, and Tricross did not differ, neither did SE and NE×SE. Cholesterol showed values between 83.87 mg/dL (SE) and 164.32 mg/dL (NE) (p < 0.05), and NE, SE, and Tricross differed from all other groups, but NE×AN and NE×SE did not differ. For triglycerides, mean values were observed between 15.80 mg/dL (NE×SE) and 28.55 mg/dL (NE) (p = 2.7197 × 10−9), and SE, NE×AN, and Tricross groups did not differ. Serum glucose concentration ranged from 91.40 mg/dL, Tricross to 161.03 mg/dL, NE (p = 1.1544 × 10−11), with no significant difference between SE, NE×AN, and Tricross groups, while the NE×SE group differed from all the others. The total bilirubin value for the groups was between 0.21 mg/dL (NE) and 0.32 mg/dL (SE) (p = 1.65 × 10−3), the NE×AN, NE×SE, and Tricross groups did not differ from each other or the purebred NE and SE groups. Direct bilirubin varied between 0.08 mg/dL (NE) and 0.26 mg/dL (SE) (p = 0); the NE×AN, NE×SE, and Tricross groups did not differ from each other or the purebred groups. Indirect bilirubin showed values between 0.06 mg/dL in SE and NE×SE and 0.13 mg/dL in NE (p = 5.5914 × 10−7), while NE and Tricross did not differ, nor did the others. Average values are described in Serum creatinine concentration showed mean values from 1.24 mg/dL in NE×AN to 1.53 mg/dL in NE and Tricross groups (p = 0.319), with no significant difference between groups. The ionized calcium concentration showed values between 4.38 mmol/L in Tricross and 4.60 mmol/L in SE (p = 0.059), with no significant difference between the groups. Serum chloride was between 99.25 mmol/L (SE) and 160.08 mmol/L (NE×SE) (p = 0.229), with no significant difference between groups. Serum iron showed values between 111.71 µg/dL (NE) 160.08 µg/dL (NE×SE) (p = 7.9115 × 10−7), while NE and Tricross, and SE and NE×SE groups did not differ, and NE×AN differed from all with intermediate value. Serum potassium concentration ranged between 3.78 mmol/L (NE×AN) and 4.15 mmol/L (SE) (p = 0.0135), with only NE×AN differing significantly from the others. For serum magnesium, there were mean values between 1.97 mg/dL (SE) and 2.80 mg/dL (NE) (p = 0), while NE×SE and Tricross did not differ, and NE×AN differed from the others with intermediate values. For serum sodium concentration, values were determined between 138.47 mmol/L (NE×AN) and 142.40 mmol/L (NE) (p = 3.207 × 10−8); the NE and NE×SE groups did not differ, as well as SE, NE×AN, and Tricross. For serum phosphorus concentration, the established values were 6.03 mg/dL (NE×AN) and 7.06 mg/dL (NE and Tricross) (p = 9.3762 × 10−9), while SE, NE×AN, and NE×SE groups did not differ.In general, serum electrolyte concentrations had little variation between groups, and the average values are in p = 9.7105 × 10−9), while NE, SE, and NE×AN did not differ with intermediate values. For serum albumin, the mean values varied between 2.92 g/dL (SE) and 3.38 g/dL (NE) (p = 0); NE and Tricross did not differ, nor did NE×AN and NE×SE. The mean values obtained for ceruloplasmin concentration are between 9.3 mg/dL (NE×SE) and 13.4 mg/dL (SE) (p = 0.013), while the NE, NE×AN, and Tricross groups did not differ from each other, without differing from the extremes. Transferrin demonstrated mean values between 72.5 mg/dL (Tricross) and 129.2 mg/dL (SE) (p = 2.6434 × 10−13); the Tricross and NE×AN groups did not differ, nor did NE×SE and NE. For haptoglobin, the observed values ranged between 22.1 mg/dL (NE×AN) and 30.6 mg/dL (Tricross) (p = 1.466 × 10−5), and the NE and Tricross groups did not differ, nor did SE and NE×SE. As for α-1-acid glycoprotein (α1GA), the values were 14.3 mg/dL (NE×AN) and 24.2 mg/dL (Tricross) (p = 3.882 × 10−5), with SE and Tricross not differing from each other, as well as NE and NE×SE; however, the latter also did not differ from the extremes. Total serum protein concentration showed values between 5.70 g/dL (NE×SE) and 6.41 g/dL (Tricross) , Finally, the RIF analysis was significant  for Fe, Creat, Cpl, and PLT, which can be considered as key metabolites for impacting global difference between the NE group and the SE group, while Tricost and Σ (epg) are responsible for the global difference between SE and NE.The RBC value showed significant differences only between the Nellore and Senepol breed groups, the crossbreds being similar to pure breeds. Ref.  observedAccording to ,43, RBC The NE group proved to be more responsive to all stimuli considered, corroborating the results of ,45. TaurHt, MCV, and TPC values showed no difference between groups, and their results are similar to those obtained by  in NelloThe higher BrW values for the purebred, NE, and SE groups are credited with the fact that these animals are the product of in vitro fertilization since the oocytes that produce such fetuses are exposed to cell growth factors originating from the maturation and in vitro fertilization media, inducing greater fetal growth and eventually overgrowth syndrome .The crossbred groups of Senepol, NE×SE, and Tricross showed higher ADWG than the other groups. Such a difference can be credited to the hybrid vigor by crossing zebuine and taurine breeds, corroborating with  and by tThe higher MDWG of SE animals is consistent with the descriptions of , who desRegarding the evaluation of gastrointestinal parasite infestation, no differences were observed in the levels of infestation between the groups NE, NE×AN, NE×SE, and Tricross by the different families of parasites, which can be credited to the zebu heritage ,66,67. TLymphocytes are the dominant cell type in the bovine species, and their count varies with age . The meaAccording to ,72, it cBasophils, in general, are responsible for modulating hypersensitivity reactions, inflammatory and autoimmune disorders, and cancer . TherefoThe activity of serum enzymes ALT, CK, and GGT did not differ between the experimental groups. There were, however, only two points of divergence between the groups. Firstly, the NE×AN group showed lower AST dosage compared to the other groups. According to , the eleSecondly, as for serum ALT activity, Refs. ,83 pointAccording to , there wThe set of metabolic variables studied showed agreement with the results for the species in different age groups obtained ,82,88,88The NE group presented higher mean values for cholesterol, glucose, and triglycerides, energy metabolites in which serum concentrations are altered by cortisol levels; that is, they increase according to the submission to stressful stimuli . Such inAn inversion between the mean values of indirect and direct bilirubin was only observed in the NE group, which contradicts the results of the other groups and is not described in the literature.There were a few differences between the groups for the electrolytic variables, which were also observed in the set of metabolic variables and the serum enzymatic activity. These point differences, such as in levels of P, Na, Mg, K, Fe, AST, total protein concentration (TP), creatinine, and albumin, can be diluted with a larger number of samples, whereas works with similar objectives to these have larger sample groups, as in , with 36The results obtained do not differ from those published by  in adultAccording to , helmintThe concentration of total serum proteins corresponds to the sum of serum albumin, α-, β-, and γ-immunoglobulins and APPs . Lower vThe mean values of TP and albumin of the NE and Tricross groups follow the results obtained by  in GuzerRhipicephalus (Boophilus) microplus ticks, decreasing appetite and leading to reduced weight gain, which may relate to the lower hardiness of taurine breeds. Parallel to the depression of appetite, these animals mobilize greater amounts of amino acids and energy for the synthesis of pro-inflammatory molecules, which reduces their efficiency within production systems where the challenge by pathogens and parasites is greater [The concentrations of APPs in the SE, NE×AN, and NE×SE groups proved to be higher compared to the pure zebu, NE, due to their greater inflammatory response to these parasitic agents, corroborating with  in lacta greater .Therefore, it can be concluded that Zebu animals and some of their more rustic crosses tend to have higher levels of TP due to the higher concentration of albumin and lower concentrations of APPs due to their lower susceptibility to diseases and, therefore, greater rusticity, while opposite behaviors are observed in taurine breeds and their crosses less resistant to stress factors.Ceruloplasmin (Cpl) is a protein produced by hepatocytes carrying copper (40 to 70% of plasma copper) . Its maiAmong the experimental groups, there was little difference in the values of Cpl concentration. SE showed higher Cpl concentrations, which may relate to the lower hardiness inherent in taurine breeds, accompanied by the NE×AN and Tricross groups. However, the NE×SE group showed a lower value than the others and is statistically similar to the value of the NE group, which suggests that this group has significantly inherited the hardiness of the Nellore and Senepol breeds, which, despite being taurine, is recognized for both. Ref.  pointed Ref.  designatTransferrin (Tf) is an APP responsible for chelating free iron, making it unavailable for microbial metabolism . The incAmong the groups, the purebreds NE and SE showed higher serum Tf concentrations, which may confer greater resistance to microbial diseases to zebuine breeds and African taurines, such as N’Dama, that contributed to the formation of the Senepol breed. The crossbred groups, NE×AN, NE×SE, and Tricross, showed lower results than the purebred breeds: NE×SE showed higher values than the groups with a breed percentile of Aberdeen Angus, a British taurine breed of known superior susceptibility to microbial diseases. No baseline values for serum Tf concentration were found in the species, but Refs.  and 116116 pointHaptoglobin (Hp) is a very important major expression pattern APP for ruminant species . Its funHowever, the results of the experimental groups may not coincide with published descriptions of studies with sick animals. The NE group showed higher baseline values, followed by Tricross, SE, NE×AN, and NE×SE, respectively. Knowing the lower susceptibility to disease of zebuine animals, it can be suggested that they present higher basal values, but their response to infection corresponds to that described by ,121.The mean values observed are similar to those obtained by  in adultThe α1GA, on the other hand, has anti-inflammatory and immunomodulatory actions, mainly inhibitory for neutrophils and the complement system, and maintenance of vascular permeability , transpoThe SE, NE×SE, and Tricross groups showed higher serum α1GA concentrations, while NE×AN showed the lowest value and NE intermediate to the others. This behavior may indicate that the Senepol breed has a less intense inflammatory response, which is transmitted to its crossbreeds. This trait may have an opposite behavior about the Aberdeen Angus breed since its double-cross breed with Nellore showed a lower average value. The mean values for serum α1GA concentration of the experimental groups are similar to those observed by  in tauriThe global distance of the mean values between the NE and SE groups shows that a pure breed stands out for its rusticity, NE, and the other, SE, for its productive potential. Considering the extensive production system applied in the Brazilian savannah region and the NE breed available , combineThe trend between the averages shows that NE×AN and Tricross are closer and, therefore, tend to present closer results. Even though Tricross has superior MDMG than all groups, which is credited to the milk production potential of its NE×AN bi-crossbred hand , it is aNE has lower levels of Cpl; however, it also shows lower serum concentrations of Fe, which may contribute to its greater resistance to infectious processes. Additionally, the concentration of serum Fe may be under the influence of Tf, which also has a regulating function of serum iron levels , since tFor the SE group, the mean values of Tricoss. (epg) and Σ (epg) were responsible for distancing from the others. Being a pure taurine breed, such results were not surprising, considering the breed group’s greater susceptibility to parasite infestation ,66,67.Most hematological variables, serum biochemistry, and APPs concentrations were not influenced by breed. However, temperament and parasitic infestation produced effects in some laboratory tests, which generated differences between breeds. There is a consistent literature record on the effect of age, mainly on hematological data. However, the repetition of the analyses with a larger sample universe, staggered at various moments with the same experimental units, may show differences compatible with those demonstrated in the literature considering the alterations in the variables studied due to age, corroborating the literature that deals with older animals. Taking together the present results, we suggest that parasite resistance may be a key factor in adaptation to the Brazilian savannah. In conclusion, all breed types evaluated in the work were healthy and had a favorable potential for production. The level of technicality of activity in the herd will decide which breed type is more suitable, more rustic, resistant to parasites, and therefore more productive.


Foliar fertilizers were applied twice, at phenological stages of beginning of pod development and grain filling. Grain yield increased with the application of MNF, yet Se rates increased Se contents linearly up to 80 g Se ha−1, regardless of the use of MNF. Lança and M5917 genotypes had grain Se critical thresholds of 1.0 and 3.0 mg kg−1, respectively. The application of Se favored higher contents of K, P, and S in grains of genotype Lança and higher contents of Mn and Fe in grains of genotype M5917. Our findings highlight the importance of addressing different Se fertilization strategies as well as genotypic variations when assessing the effects of Se on soybean yield and grain quality.Selenium uptake and its content in soybean grains are affected by Se application methods. This study evaluated the impact of Se foliar application combined with a multi-nutrient fertilizer (MNF) on soybean, establishing a Se threshold to better understand the relationship between Se content in grains and yield of two genotypes (58I60 Lança and M5917). Two trials were conducted in a 4 × 2 factorial design: four Se rates (0, 10, 40, 80 g Se ha Soybeanpercent) ,35. As aThe results obtained showed a great influence of the genotypes for most of the analyses performed . They wep > 0.05). On the other hand, MNF application influenced the yield of both genotypes (p < 0.05)  for 58I60 Lança and M5917 genotypes, respectively, compared with the application of Se without MNF. In general, the genotype M5917 showed the highest grain yield—5.0 t ha−1 on average—while the genotype 58I60 Lança produced 4.2 t ha−1. Average yields with the MNF application varied from 4.3 to 4.7 t ha−1 for genotype 58I60 Lança, and from 5.0 to 5.4 t ha−1 for genotype M5917.The spraying of Se rates did not affect grain yield ( < 0.05) A,B. The −1), regardless of the use of MNF. In 58I60 Lança, the increase in grain Se content for each gram of Se applied via foliar was 0.063 mg kg−1 with MNF and 0.055 mg kg−1 without MNF. In genotype M5917, the Se content in grain increased by 0.087 mg kg−1 with MNF and 0.065 mg kg−1 without MNF for each gram of Se applied through foliar supply. According to the confidence interval (95%), the application of MNF did not affect the Se content in the soybean grains up to the rate of 43.8 g Se ha−1 for genotype 58I60 Lança and 29.4 g Se ha−1 for genotype M5917. However, after these Se rates, the association of Se with MNF promoted higher Se content.Selenium content in grains was affected by the interaction between Se rates and MNF application A,B. In bp < 0.05) , 31.1% and 37.7%, respectively. The Se recovery obtained with the application of Se foliar at 10 g Se ha−1 was 21.5%.The Se recovery rate (%) by soybean grains was also affected by Se foliar rates ( < 0.05) C,D. The p < 0.05)  in the 58I60 Lança genotype ( < 0.05) . Despitep < 0.05). The content of Mg, Ca, and Fe was not affected by the factors studied (p > 0.05). Following in the genotype M5917, the K content in the grains fitted quadratic regression in the levels of Se supplied via foliar application (R2 = 77%). The highest K content in grains was 18.57 g Se ha−1 and it was obtained by the application of 45.6 g Se ha−1. For genotype M5917, the F test for Cu and Zn showed significant differences among Se foliar rates and MNF . On the 2 = 63%), and the highest S content was obtained by the rate of 37 g Se ha−1.Concerning genotype 58I60 Lança, the S content increased linearly upon increasing the Se rate, regardless of the MNF application, with a correlation coefficient of 89% being observed with MNF and 68% without MNF. The MNF promoted a higher accumulation of S in grains, regardless of the Se rate in genotype M5917. The S data were fitted to a quadratic model (Rp > 0.05)  . The tot−1 and 3.0 mg kg−1 for genotypes Lança and M5917, respectively . As per the estimated daily Se intakes shown in −1) would be: 15.1 g Se ha−1 (with MNF—genotype 58I60 Lança), 16.2 g Se ha−1 (without MNF—genotype 58I60 Lança), 15.3 g Se ha−1 (with MNF—genotype M5917), and 15.4 g Se ha−1 (without MNF—genotype M5917).−1 in grains were 19.5 g Se ha−1 (with MNF) and 23.3 g Se ha−1 (without MNF) for the Lança. In the genotype M5917, the rates to produce soybean grains with 3 mg kg−1 of Se are 36.1 g Se ha−1 (with MNF) and 49.3 g Se ha−1 (without MNF).Considering the daily Se consumption by person, the adequate Se rates to produce enriched soybean grains are below the Se content that reduces crop yield . Indeed, using the model fitted for Se content in grains A,B, Se r2 = 65%), P (R2 = 57%), and K (R2 = 57%) content in grains. For genotype M5917, the increase of Se in the grains increased Mn content (R2 = 44%), and Fe (R2 = 51%). Selenium content in grains negatively affected yield and total free amino acid content in the genotype Lança . This effect was not observed for cultivar M5917.The correlation between the variables assessed is presented in Selenium is not considered a nutrient for plants, yet its beneficial effects are already well established ,36. The −1 for Lança and 29.4 kg Se ha−1 for genotype M5917, which is about 33% less, indicating that the application is more efficient for the last genotype. This also demonstrates that the interaction of Se foliar and other nutrients can enhance the uptake/redistribution of Se in plants. Foliar spraying with Se resulted in a linear increase of the Se content in soybean grains A,B. SimiNitrogen is one of the components of MNF and it is known that it can affect Se uptake by plants . The facPrevious studies have produced conflicting findings regarding the response of various essential elements to Se treatment in several crops. In rice, Se application combined with N fertilization resulted in higher grain concentrations of N, P, K, Ca, Se, B, Cu, Zn, and Mo . Wheat cSeveral factors influence selenium recovery, including the Se source and the plant species. In a previous study carried out by our research group, the recovery of Se applied via soil was 6.5% for genotype M5917 and 8.1% for genotype Lança . In the Changes in the content of the nutrients in grains are shown in −1 of Se in grain, the yield in the genotype 58I60 Lança is reduced, while genotype M5917 showed this effect only above 3.0 mg Se kg−1, which is a clear indication that genotype M5917 is more tolerant to Se accumulation in grains than 58I60 Lança. Selenium toxicity in plants has not been fully investigated, but it has been demonstrated that tolerance varies depending on plant species and genotypes [The critical Se threshold demonstrated that above 1.0 mg kgenotypes . Indeed,enotypes , with th−1 for wheat, 41.5 mg kg−1 for rice, 76.9 mg kg−1 for corn, and 104.8 mg kg−1 for brown mustard [In another study assessing Se toxicity to various crops, brown mustard has shown a greater tolerance to Se when compared with maize, rice, and wheat . From th mustard .−1, may lead to toxicity, resulting in health problems. Some of the Se toxicity symptoms are hair and nail loss, skin lesions, nervous system dysfunction, and even death [As seen in our study, Se intake values for biofortifying soybean, considering the selected data for adequate daily intake and average consumption of soy protein, are lower than the level of Se in grains that reduced yields C,D. Thisen death ,54. As e2O) = 6.0; H + Al = 2.96; Al = 0.06; P (Mehlich-1) = 34.8 mg dm−3; K = 148 mg dm−3; S = 4.11 mg L−1; CEC = 9.83 cmolc dm−3; Ca = 5.05 cmolc dm−3; Mg = 1.44 cmolc dm−3; P-rem = 28.10 mg L−1; organic matter = 2.69 dag dm−3; clay = 510 g kg−1; silt =110 g kg−1; and sand =380 g kg−1.Two similar trials were conducted on a soybean field during the 2018/2019 cropping season, at Farm Uva, municipality of Capão Bonito, São Paulo state, Brazil , with an average annual rainfall of 1628 mm and an average annual temperature of 18.8 ºC, under a humid subtropical climate (Cfa) . The soi−1) and two methods of Se application: (i) Se with multi-nutrient fertilizer  and (ii) Se without MNF. The experiments were composed of four blocks, totalizing 32 experimental plots for each genotype. The treatments with MNF received 2 kg ha−1 of the product; its composition was as follows: nitrogen (N—5%), phosphorus pentoxide (P2O5—10%), potassium oxide (K2O—20%), magnesium (Mg—29%), sulfur (S—12%), and boron (B—0.5%). Sodium selenate was used to prepare the Se solution . Foliar spray fertilizers were applied twice, at phenological stage R3 (beginning of pod development) and R5 (grain filling), with each application containing half of the total dose. Sodium selenate and MNF (if used) were diluted in deionized water, mineral oil was added, and a pressured carbon dioxide pump was used to apply it.Each experiment was carried out with one soybean genotype, 58I60 Lança, and M5917. The experiment was arranged in a randomized block with a full factorial design of 4 × 2, being four Se rates , considering 13% moisture. After that, the grains were dried until they reached a constant weight and were ground with an electric hand mill. The grains from the useful area of each experimental plot were harvested after maturity and soybean yield was calculated by weighing the grains and extrapolating to a hectare (kg ha3 (65%) was added. The extract was allowed to stand at room temperature overnight before being digested the next morning (16 h). The vessels were then hermetically sealed and heated in a Mars-5 microwave digestion oven  following the 3051A methodology, proposed by the United States Environmental Protection Agency [To determine Se, macronutrients, and micronutrients, 0.5 g of samples were placed in Teflon vessels and 5 mL of HNOn Agency . The con−1 of pure Se . To maintain digestion quality, each batch of digestion included standard reference material from the Institute for Reference Materials and Measurements  and a blank sample. The main recovery value for standard material was 95% (n = 5). The detection limit (LOD) was calculated by taking the standard deviation and mean of 7 blank extracts. Selenium content in the solutions was measured using a graphite furnace atomic absorption spectrometer . The calibration curve for Se measurement was obtained from a standard solution containing 1 g kgThe fraction of the applied Se incorporated in soybean grains (Se recovery) was calculated using Equation (1) described below:−1) = Se content in soybean grains from soybean plants grown in treatments that had received Se applications, considering the yield obtained in each treatment; Se treatment (g Se ha−1) = Se content in soybean grains from soybean plants grown in treatments without Se applications, considering the yield obtained in each treatment;−1 day−1) = estimated daily Se intake per person; Se control (g Se ha−1) = the amount of selenium in soybean grains; Se (µg kgProt (g) = the average amount of protein in soybean grains (42.4 to Lança and 41.8 to M5917). The ninhydrin method was used to determine total free amino acids . The prop < 0.05) for grain yield and Se recovery analysis. Pearson’s linear correlation matrix (p < 0.05) was also used to validate clusters and potential relationships of Se rates applied and plant attributes. The R software was used to carry out the analyses [The obtained data were analyzed for normality (Shapiro–Wilk test) and variance homogeneity (Bartlett’s test). They were then analyzed using analysis of variance (ANOVA), and when significant, linear, or quadratic regression models were fitted. The linear models were compared to Se content in grains, using a confidence interval, created at 95% of probability. The treatments were compared using the Tukey test . The yield of soybean present in the database was converted into relative yield (%) by taking into consideration each genotype and crop season to develop critical threshold estimation models. Models were developed on the boundary line ,62 using−1), regardless of the use of MNF. The Lança and M5917 genotypes of soybean grains had Se critical thresholds up to 1.0 mg kg−1 and 3.0 mg kg−1, respectively. The application of Se increased the content of K, P, and S in the grains of the genotype 58I60 Lança as well as the content of Mn and Fe in the grains of the genotype M5917. Finally, we recommended applying 15.1 g Se ha−1 to genotype 58I60 Lança and 15.3 g Se ha−1 to genotype M5917, combined with MNF to improve the Se content in the grains. Se supply through foliar application in soybean plants is a notably good strategy to improve intake of Se by the population, and an association with other nutrients can improve the efficiency of biofortification strategies. Despite this, Se foliar applications should be used cautiously to avoid Se toxicity and yield loss in plants. The grain yield was higher with MNF application. In both genotypes, the Se rates increased the Se content linearly up to the highest rate (80 g Se ha


Pesticides are toxic and could negatively impact humans and the ecosystem. The Kitchener Drain is among the longest drains in Egypt and carries a wide range of wastewater from the agriculture sector, which contains pesticides and may pollute the ecosystem. Thus, water quality, human health risk, and pesticide accumulation in African catfish and Nile tilapia from the Kitchener Drain-Egypt. The water and fish samples were collected from Kitchener Drain in Kafr Elsheikh Governorate, Egypt, during the four seasons. The results indicated that heptachlor and diazinon were undetected during the four seasons. However, endosulfan, chlorpyrifos, and dicofol were detected in winter and autumn. Only p,p′-DDT was detected during spring. Endosulfan, heptachlor, and aldrin were detected in Nile tilapia during winter. Only heptachlor and aldrin were detected during spring. Endosulfan, heptachlor, dicofol, p,p′-DDT, chlorpyrifos, and diazinon were detected in the autumn season. In summer, dicofol and p,p′-DDT were detected, while endosulfan, heptachlor p,p′-DDT, aldrin, chlorpyrifos, and diazinon were not detected. In African catfish, endosulfan, heptachlor, dicofol, and p,p′-DDT were detected during winter, while chlorpyrifos, aldrin, and chlorpyrifos, aldrin, and diazinon were not detected. In the spring season, endosulfan, heptachlor, and aldrin were detected. Endosulfan, heptachlor, dicofol, p,p′-DDT, aldrin, chlorpyrifos, and diazinon were detected in the autumn season. Similarly, in the summer season, endosulfan, heptachlor, dicofol, p,p′-DDT, aldrin, chlorpyrifos, and diazinon were detected. The sequence of estimated daily intake (EDI) in Nile tilapia during the four seasons is heptachlor > endosulfan > dicofol > p,p′-DDT > aldrin > diazinon > chlorpyrifos. The sequence of EDI in African catfish during the four seasons is endosulfan > p,p′-DDT > heptachlor > aldrin > dicofol > diazinon > chlorpyrifos. In conclusion, the results confirmed the absence of a hazard index for consuming Nile tilapia and African catfish collected from the Kitchener drain. Directly or indirectly, these toxicants' derivatives reach the water bodies and induce negative impacts on the ecosystem2. Many studies tackled the impacts of accumulated pesticides and their derivatives on water drains, sediments, and living organisms, especially fish3. The hazards of water pollution with pesticides affect human health since accumulated derivates could reach the human body through the water or fish as food4. Fish are utilized as bioindicators in case of high pollution in the water bodies due to their low digestion capacity for the accumulated pesticides7. Pesticides and toxicants can accumulate in the fish's body through the gills, skin, and intestines8.A considerable number of pesticides and insecticides are used in the agriculture sector9. Organochlorine and organophosphorus pesticides are heavily used in agriculture, and their derivates could pollute the water bodies and cause severe impacts10. Besides, organochlorine and organophosphorus compounds are known for their toxic effects on humans and the ecosystem due to their long persistence, high bioaccumulation capacity, and long-range transport9. Further, the sediments and some fish species, such as Nile tilapia and African catfish, are rich in organochlorine and organophosphorus derivates and may have human health risk12. One of the famous drains in the northern area of Egypt is the Kitchener drain which collects water from the agricultural and industrial sectors13. More specifically, the existing section of the Kitchener drain in Kafrelsheikh governorate, which is famous for agriculture and fish farming activities. Nile tilapia is a known species worldwide and can grow under farming conditions or in the wild14. Another well-known fish species, famous for its delectable taste, is the African catfish which prefers to live in the bottoms of water bodies. This results in a high possibility of organochlorine and organophosphorus accumulation through the sediments15. Both fish species thrive in the Kitchener drain and can be captured to be introduced into the human food chain. Therefore, the human food chain is expected to get polluted with pesticide derivates through the water and fish. Hence, this study was conducted to test the presence of organochlorine and organophosphorus in the Kitchener drain using Nile tilapia and African catfish as bioindicators. Further, the pesticides were tested in the muscles of Nile tilapia and African catfish, as well as the human risk assessment.Pesticides are known for their toxic effects on humans and the ecosystem due to their long persistence, high bioaccumulation capacity, and long-range transport16. Egypt is famous for agricultural activity, which depends on using a wide range of pesticides. Nevertheless, enormous amounts of pesticides threaten the water bodies, and consequently, their waste pollutes the drainage water17. Kitchener drain is a central drain that crosses through several vital crowdy cities in the Delta area. Its water is full of pesticides and industrial waste that can reach the human food chain and cause severe health risks18. The aquatic system is also expected to suffer from the pollution of pesticides that may accumulate in the fish's edible tissues19. The bioaccumulation of pesticides was detected earlier in some famous consumed fish species in Egypt, such as Nile tilapia and African catfish12. Hence, this study tested the presence of organochlorine and organophosphorus in the Kitchener drain using Nile tilapia and African catfish as bioindicators. Further, the pesticides were tested in the muscles of Nile tilapia and African catfish and the human risk assessment.Pesticide pollution is increasingly spreading, especially in developing countries, due to the lack of awareness and regulationsThe Kitchener Drain is among the longest in Egypt, with a total of 69 km, and passes through three governorates in the Delta area . Forty-six kilometers of the Kitchener Drain is located inside Kafr El-Sheikh governorate before draining the water to the Mediterranean Sea. El-Burullus lagoon and surrounding areas located in Baltim city in Kafr El-Sheikh are famous for aquaculture-related activities where the water in Kitchener Drain is used for fish farming.Oreochromis niloticus) and African catfish (Clarias gariepinus)) were collected. Fish was collected and kept in an ice box at 4 °C. Subsequently, water samples were collected 20 cm below the water surface and kept in sterile water containers while treated with 1 mL of HCL to eliminate microbial activity.The samples were collected from Kitchener Drain in Kafr Elsheikh Governorate, between longitudes 31° 08′ 25.6′′ E and latitudes 31° 18′ 02.4′′ N  to remove suspended materials. Water samples were collected and kept in 500 mL sterile plastic containers. After filtration, samples were stored in dark glass vials for GC determination. Water samples were divided into two parts, the first part for determining the water’s physical parameters and the second part for analysis of pesticides.20 to analyze semi-volatile and non-volatile organic compounds. Water samples (500 mL) were transferred into a 1.000 mL separatory funnel. The samples were extracted thrice with 100 mL portions of 1:1 (v/v) ethyl acetate dichloromethane. The separatory funnel was clapping for 30 min to allow phase separation. The combined organic phases were collected into a 500 mL beaker, with the aqueous phase discarded. The combined organic layer was dried from the aqueous substance with 20 g of anhydrous sodium sulfate and allowed to settle. The organic content was decanted into a 300 mL round bottom flask, and the content was evaporated to dryness using the rotary evaporator at 40 °C. The pesticide was dissolved and collected with 2 mL of ethyl acetate and transferred into a 2 mL vial to be ready for cleanup21.The water samples were prepared and analyzed sequentially for endosulfan, heptachlor, dicofol, p,p′-DDT, aldrin, chlorpyrifos, and diazinon according to the extraction technique for aqueous matrix was carried out according to Edgell and Wesselman22.Two fish species (Nile tilapia and African catfish) were caught from the Kitchener drain during the four seasons Table . Twelve The estimated daily (EDI) or weekly intake (EWI) of endosulfan, heptachlor, dicofol, p,p′-DDT, aldrin, chlorpyrifos, and diazinon by fish consumption were examined using the two equations below:23.where, Cm represents the oregano chlorine concentrations in fish samples (μg/g-ww); IR is the daily intake rate of fish (62.25 g/person/day) and BW is the average body weight EF: the exposure frequency (days/year); ED : the exposure duration-years; IR: the ingestion rate -g/day; C: the metal concentration in fish (μg/kg); RfD: the oral reference dose  mg/kg/day; WAB: the average (kg) body weight, and ATn : the average exposure time (days/year × ED) for non-carcinogens.24.The hazard index (HI) has been performed to assess the probable human health hazard of organochlorine and organophosphorus compounds. The HI refers to the sum of all THQ for various organochlorine and organophosphorus compounds exposures as shown in the equation below:The authors are informed and agree to the study.The ethical committee of the Faculty of Aquatic and Fisheries Sciences, Kafrelsheikh University approved the experimental protocol and all methods in the present study for treating animals for scientific purposes. All experiments were performed in accordance with relevant guidelines and regulations. Our reporting of research involving animals follows the recommendations of the ARRIVE guidelines.15, who stated that the levels of endosulfan (18.6–40 ppb), heptachlor (22.8–47.1 ppb), dicofol (17.4–31.8 ppb), p,p′-DDT (25.8–88.3 ppb), and chlorpyrifos (37–53.8 ppb) were detected in the water samples collected from the river Nile during the four seasons. According to the European Water Framework Directive 25, the detected pesticide levels in this study are not significantly harmful and toxic. Several factors affect the drainage water in big drains, such as the effluents of agricultural and industrial wastes and untreated sewage water26. Kitchener Drain receives drainage from several agricultural drains containing pesticides and insecticides13. Organochlorine and organophosphorus based pesticides are heavily used in agriculture, but their derivates could pollute the water bodies and cause severe impacts10. The pesticides are applied in the agriculture sector, where the water runoff can affect the dilution of organochlorine and organophosphorus compounds3. Indeed, soil erosion and the level of suspended derivatives, including organochlorine and organophosphorus compounds, are strongly affected by rainfall events27. Hence, the high flood rate of water during the summer results in high dilution for the organochlorine and organophosphorus compounds15 and, thereby, low presence in the water and fish samples.In this study, the water samples were collected from Kitchener Drain Table , and theTable 15 stated that pesticides were also detected in tilapia collected from the river Nile, Cairo, Egypt, during the four seasons. Generally, the levels of endosulfan showed the highest detected pesticides with a total of 50.8 ppb in Nile tilapia during the four seasons, which is within the permitted levels as indicated by FAO28. Endosulfan is commonly used in agriculture, but the high accumulated levels cause endocrine disruption involved in fish growth and development. The detected heptachlor is 43.5 ppb which is within the permitted levels as reported by FAO28 and USFDA29, while it is over the permitted levels reported by CFIA30 and FSANZ31. The total detected p,p′-DDT (31.9 ppb), and aldrin (26.23 ppb) during the four seasons are within the accepted levels as indicated by FAO28, USFDA29, CFIA30, and FSANZ31. Chlorpyrifos showed a total of 10.8 ppb during the four seasons, which is considered a safe level as indicated by FAO28, while diazinon showed a total of 17.5 ppb, which is also safe compared to the reports of FAO28, CFIA30, and FSANZ31. It is worth noting that the highest organochlorine and organophosphorus compounds have been detected in Nile tilapia in autumn (106.8 ppb), followed by winter (45.63 ppb), then summer (40.9 ppb) and spring (33 ppb), respectively. This might be as a result of the high hydrophobicity and lipophilicity of these compounds as well as their potential retention in organisms' organic phases32.In Nile tilapia, endosulfan (15.2 ± 4.15 ppb), heptachlor (16.4 ± 5.15 ppb), and aldrin (14.03 ± 2.65 ppb) were detected during the winter season. Only heptachlor (20.8 ± 4.87 ppb) and aldrin (12.2 ± 1.9 ppb) were detected in Nile tilapia during spring. In the autumn season, endosulfan (35.6 ± 6.95 ppb), heptachlor (6.3 ± 1.73 ppb), dicofol (21.3 ± 6.53 ppb), p,p′-DDT (15.3 ± 5.56 ppb), chlorpyrifos (10.8 ± 3.92 ppb), and diazinon (17.5 ± 4.53 ppb) were detected in Nile tilapia. In summer, dicofol (24.3 ± 5.92 ppb) and p,p′-DDT (16.6 ± 3.42 ppb) were detected, while endosulfan, heptachlor p,p′-DDT, aldrin, chlorpyrifos, and diazinon were not detected in Nile tilapia. In line with the current study, Shalaby et al.15 who stated that pesticides derivatives were detected in African catfish collected from the river Nile, Cairo, Egypt, during the four seasons. In the same manner as Nile tilapia, endosulfan showed the highest detected level (64.81 ppb) in African catfish during the four seasons but still within the permitted levels, as reported by FAO28. The detected heptachlor is 37.98 ppb which is within the permitted levels as reported by FAO28 and USFDA29, while it is over the permitted levels reported by CFIA30 and FSANZ31. The total detected p,p′-DDT (37.3 ppb), and aldrin (36.34 ppb) during the four seasons are within the accepted levels as indicated by FAO28, USFDA29, CFIA30, and FSANZ31. Chlorpyrifos showed a total of 8 ppb during the four seasons, which is considered a safe level as indicated by FAO28, while diazinon showed a total of 18.4 ppb, which is also safe compared to the reports of FAO28, CFIA30, and FSANZ31. The detected pesticides in African catfish followed the sequence of autumn (79.36 ppb) > summer (59.25 ppb) > winter (49.84 ppb) > spring (40.13 ppb). Shalaby et al.15 reported similar results where the pesticides residues showed the same manner (autumn > summer > winter > spring) in African catfish and tilapia collected from the river Nile, Cairo, Egypt. Both fish species are potential targets for organochlorine and organophosphorus compounds accumulation from the pesticides polluted water. In African catfish, the high-fat content allows the soluble lipids organochlorine and organophosphorus compounds to accumulate massively in edible fish tissue causing severe pollution.In African catfish, endosulfan (19.21 ± 3.95 ppb), heptachlor (10.93 ± 1.68 ppb), dicofol (9.6 ± 1.23 ppb), and p,p′-DDT (10.10 ± 1.63) were detected during the winter season while chlorpyrifos, aldrin, and diazinon were not detected. In the spring season, endosulfan (21.3 ± 3.95 ppb), heptachlor (10.6 ± 1.51 ppb), and aldrin (8.23 ± 3.1 ppb) were detected in African catfish. In the autumn season, endosulfan (10.70 ± 2.65 ppb), heptachlor (13.85 ± 2.64 ppb), dicofol (5.5 ± 1.71 ppb), p,p′-DDT (21.50 ± 6.54 ppb), aldrin (16.51 ± 4.96 ppb), chlorpyrifos (4.70 ± 0.7 ppb), and diazinon (6.6 ± 0.073 ppb) were detected in African catfish. Similarly, in the summer season, endosulfan (13.60 ± 3.61 ppb), heptachlor (2.60 ± 0.7 ppb), dicofol (10.65 ± 2.85 ppb), p,p′-DDT (5.70 ± 0.83 ppb), aldrin (11.6 ± 4.18 ppb), chlorpyrifos (3.30 ± 0.02 ppb), and diazinon (11.80 ± 1.31 ppb) were detected in African catfish. The analyzed pesticides are in line with Shalaby et al.33. Hence, this study evaluated the estimated daily intakes (EDI) of pesticides in Nile tilapia and African catfish collected from the Kitchener drain. Several organizations report the permissible daily intake (PDI) of pesticides in fish to compare with EDI and predict the possibility of human risk35. The data in Table 34 and FDA35 (μg/kg bwt). Further, the results are similar to Barakat et al.36 and Shalaby et al.15, who stated that the EDI of Nile tilapia collected from Lake Qarun and Nile River in Cairo were not over the PDI.The accumulation of pesticides in fish affects the food chain through daily intake34 and FDA35. Similarly, Shalaby et al.15 stated that the EDI of African catfish collected from the Nile River in Cairo was not over the PDI.In African catfish, the EDI of endosulfan (0.009–0.018 μg/kg BW/day), heptachlor (0.002–0.012 μg/kg BW/day), dicofol (0–0.009 μg/kg BW/day), p,p′-DDT (0–0.019 μg/kg BW/day), aldrin (0–0.014 μg/kg BW/day), chlorpyrifos (0–0.002 μg/kg BW/day), and diazinon (0–0.01 μg/kg BW/day) during the four seasons. Nevertheless, the EDI during the winter  and during the spring  recorded 0 μg/kg BW/day. The sequence of EDI in African catfish during the four seasons is endosulfan > p,p′-DDT > heptachlor > aldrin > dicofol > diazinon > chlorpyrifos. The results also indicate that the EDI in African catfish collected from the Kitchener drain during the four seasons is lower than the PDI37. In addition, the consumed pesticides in agriculture is a season-dependent practice that may affect the presence of specific pesticide residuals in the surface waters38.In Nile tilapia or African catfish, the differences in the EDI during the four seasons can be altered by the surface runoff of water and levels of suspended pesticides. Concisely, the precipitation rate of pesticides may differ as a response to seasonality effects on the surface water runoff during rainy and dry seasons39. In this study, the THQ for Nile tilapia and African catfish collected from Kitchener drain less than one in the case of all detected organochlorine and organophosphorus compounds during the four seasons  refer to the possible health risk for the pesticides, where a value over one indicates health riskns Table . BesidesThe results concluded that endosulfan, heptachlor, dicofol, p,p′-DDT, aldrin, chlorpyrifos, and diazinon were detected in the water of Kitchener drain but in a season-dependent manner. It is worth noting that the highest organochlorine and organophosphorus compounds have been detected in Nile tilapia in autumn, followed by winter, then summer and spring. In addition, organochlorine and organophosphorus compounds were detected in African catfish following the sequence of autumn > summer > winter > spring. The estimated daily intake for all detected organochlorine and organophosphorus compounds was below the referenced permissible daily intake in Nile tilapia and African catfish. Besides, the results confirmed the absence of a hazard index for consuming Nile tilapia and African catfish collected from the Kitchener drain. However, future studies are suggested to confirm the results obtained by considering having more sampling sites along the Kitchener drain.