Existing literature on the association of electronic screen use duration with depression among adolescents is contradictory. The current study aimed to elucidate the association between duration of electronic screen use for non-educational purposes and depression symptoms among middle and high school students in Zhejiang Province, China.A cross-sectional study of 27,070 students in grades 7–12 from 376 middle and high schools was conducted through an anonymous self-administered questionnaire between April and June 2022. Poisson regression was utilized to examine the association between electronic screen use duration for non-educational purposes and depression symptoms.Of the 27,006 eligible students, 51.6% (13932) were boys and the mean (SD) age was 15.6(1.7) years. The overall prevalence of symptoms of depression was 22.4% (95%CI 21.4–23.4); girls  had a higher prevalence than boys . After adjustment for socio-demographic status, lifestyle factors, self-perceived health, academic performance, loneliness and sadness, compared to those who did not use electronic screens for non-educational purposes, the prevalence ratios (PRs) for depression symptoms were 1.03 (95% CI 1.02–1.04) for those exposed to electronic screens for <1 h/day, 1.07 (1.05–1.09) for 1.0–1.9 h/day, 1.10 (1.07–1.13) for 2.0–2.9 h/day, 1.14 (1.10–1.18) for 3.0–3.9 h/day, 1.18 (1.12–1.23) for 4.0–4.9 h/day, and 1.21 (1.15–1.29) for ≥5 h/day.Duration of electronic screen use for non-educational purposes was positively associated with symptoms of depression among middle and high school students, even with a relatively short daily duration of use. Mental disorders are increasingly recognized as leading causes of disease burden . The estWith rapid development of economies and technology, the variety of electronic devices available has increased dramatically over recent decades, to include not only traditional televisions and desktop computers, but also smart phones, laptops, and tablet devices etc. In the United States of America, the percentage of high school students using electronic screens for longer than 3 h per day increased from 22.1% in 2003 to 46.1% in 2019 , 6, exceHowever, existing literature on the association of duration of electronic screen use with depression among adolescents is contradictory. While some studies indicated that there was a significant positive association between screen time and depression –14, one A three-stage cluster sampling design was implemented. In stage 1, 30 counties/districts were sampled randomly from all 90 counties/districts. In stage 2, 11 classes of middle school, 6 classes of academic high school, and 6 classes of vocational high school were selected randomly within each chosen county/district. In stage 3, students in all selected classes were invited to participate in the study. A self-administered anonymous questionnaire was filled in by participants in the classroom setting without school teachers’ supervision. The field survey was carried out by trained CDC staff using standardized procedures.Depressive symptoms were assessed using the Patient Health Questionnaire 9-item depression scale (PHQ-9) , widely Duration of electronic screen use for non-educational purposes was evaluated through the question: “On an average school day, how many hours do you play pad or use a smartphone or tablet computer or desktop computer for something that is not school work?” Response options included: “I do not play pad or use a smartphone or tablet computer or desktop computer or play these only for school work,” “<1 h/day,” “1.0–1.9 h/day,” “2.0–2.9 h/day,” “3.0–3.9 h/day,” “4.0–4.9 h/day,” and “≥5 h/day.”Covariates in the present study included age, sex, region, type of school, parental educational attainment, parental marital status, household income, cigarette smoking (smoking on at least 1 day in the past 30 days), alcohol drinking , physical activity, academic performance, self-perceived health, loneliness and sadness. Sadness was assessed through the question: “During the past 12 months, did you ever feel so sad or hopeless almost every day for 2 weeks or more in a row that you stopped doing some usual activities?” (Answer options: yes and no).S) were estimated using three regression models. In model 1, PRS were adjusted for age , sex (boys and girls), region , and type of school . Model 2, additionally adjusted for paternal and maternal educational attainment  and parental marital status (married and others), household income , cigarette smoking (yes and no), alcohol drinking (yes and no), physical activity , academic performance , and self-perceived health status . Model 3, additionally adjusted for loneliness  and sadness (yes and no). In multiple linear regression analyses, the exposure variable  was converted to a continuous variable. Those who chose “<1 h/day,” “1.0–1.9 h/day,” “2.0–2.9 h/day,” “3.0–3.9 h/day,” “4.0–4.9 h/day,” and “≥5 h/day” were assigned with value 0.5 h/day, 1.5 h/day, 2.5 h/day, 3.5 h/day, 4.5 h/day, and 5.5 h/day, respectively. The association between duration of electronic screen use for non-educational purposes and depression symptoms was further examined. In sensitivity analyses, a cut-point of 15 was used to define depression for evaluation of its association with duration of screen use. All statistical analyses were performed using SAS software V.9.4 . Statistical significance level was set at p value < 0.05 using a 2-sided test.Continuous variables were presented as mean ± standard deviation. Categorical variables were presented as percent and 95% confidence intervals (CI). Weighted prevalence was calculated using the PROC SURVEYFREQ procedure. Considering high prevalence of depression symptoms, modified Poisson regression was utilized to examine the associations between duration of electronic screen use for non-educational purposes and depression symptoms . PotentiN = 12,760), 27.3% , and 25.5% , respectively.A total of 28,043 students from 376 schools were invited to participate, of whom, 27,070 students were surveyed. Overall, 114 students refused to participate and 859 were absent from school on the survey day, yielding a response rate of 96.5%. Of 27,070 questionnaires, 40 were excluded due to being incomplete, 17 were excluded due to missing one of more of the nine items in the PHQ-9 questionnaire, and 7 were excluded due to missing information on duration of electronic screen use. Eventually, 27,006 students, comprising 13,932 boys and 13,074 girls, were included in the present analyses. The mean age was 15.6 ± 1.7 years. The percentages of students attending middle schools, academic high schools and vocational high schools were 47.2% . Girls  had a higher prevalence of depression symptoms than boys . Students living in rural areas  had a higher prevalence of depression symptoms than their counterparts living in urban areas . Prevalence of depression symptoms among students attending middle school, academic high school, and vocational high school was 22.1% (95%CI: 20.7–23.5), 24.6% (95%CI: 22.4–26.7), and 20.8% (95%CI: 18.9–22.6), respectively  of depression symptoms among students was 22.4% (21.4–23.4), and was 20.1% (18.4–21.9), 23.6% (21.9–25.2), and 22.7% (21.3–24.2) for students aged ≤13 years, 14–15 years, and ≥16 years, respectively  for depression symptoms were 1.03 (1.02–1.04) for those exposed to electronic screens for <1 h/day, 1.07 (1.05–1.09) for those exposed for 1.0–1.9 h/day, 1.10 (1.07–1.13) for those exposed for 2.0–2.9 h/day, 1.14 (1.10–1.18) for those exposed for 3.0–3.9 h/day, 1.18 (1.12–1.23) for those exposed for 4.0–4.9 h/day, and 1.21 (1.15–1.29) for those exposed for ≥5 h/day, respectively . There wIn sensitivity analyses, a cut-off value of 15  was used to evaluate the association of duration of electronic screens viewing with depression symptoms. After adjusting for potential confounding factors, compared to those who were not exposed to electronic screens, the PR (95%CI) for depression symptoms was 1.04 (1.03–1.06) for those exposed to electronic screens for non-educational purposes for <1 h/day, 1.09 (1.05–1.13) for those exposed for 1.0–1.9 h/day, 1.14 (1.08–1.20) for those exposed for 2.0–2.9 h/day, 1.19 (1.11–1.27) for those exposed for 3.0–3.9 h/day, 1.24 (1.14–1.35) for those exposed for 4.0–4.9 h/day, and 1.30 (1.17–1.44) for those exposed for ≥5 h/day, respectively, which was similar to the results based on a cut-off value of 10 , the risk of depression was higher among non-users , occasional users , and heavy users (>2 h/day) . AnotherAlthough the underlying processes through which screen time could be related to the development of depression are likely to be complicated, several possible hypotheses have been proposed. First, excessive screen time has been associated with unhealthy behaviors, including inadequate physical activity and poor sleep quality, which may exacerbate depression symptoms , 37. SecThe findings of the present study are of practical public health importance, and provide crucial evidence that may inform prevention of adolescent depression. First, more than one fifth of the students suffered from depression symptoms, implying that depression is common among students of this age, and more comprehensive efforts are needed to address adolescent mental health issues in China. These might include each middle and high school being equipped with at least one full-time mental health teacher, incorporating mental health education into the school curriculum, cultivating students’ capability of solving mental crisis, strengthening early screening for depression, and providing professional medical services for those with severe depression . Second,There are several limitations of the present study. First, the cross-sectional study design prohibits establishment of the temporal relationship between screen use and depression symptoms. Second, we did not collect the information on the type of electronic device used, which may be important since previous studies have indicated that the associations between screen behavior and depression varied by screen behavior types . Third, In summary, our study sheds light on the association between duration of electronic screen use for non-educational purposes and depression symptoms among middle and high school students in Zhejiang Province, China. We found that depression symptoms were prevalent, and duration of electronic screen use for non-educational purposes was positively associated with depression symptoms among students, despite short-duration of exposure. It may be appropriate to include reduced electronic screen time in targeted prevention of adolescent depression.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of Zhejiang Provincial Centre for Disease Control and Prevention. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.HW designed the study, drafted the manuscript, and analyzed the data. NL, YG, and JP collected the data. JZ and MY were involved in data interpretation. FB provided important comments on the manuscript and revised the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by Program of Zhejiang Federation of Humanities and Social Sciences (grant number 2023B059).The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Roseibium sp. strain Sym1, a strain isolated from a monoculture of a Symbiodiniaceae marine dinoflagellate, Symbiodinium linucheae, a microalgal symbiont of cnidarians. The completed genome consists of one circular chromosome of 6,694,563 bp and four plasmids of 192,102 bp, 160,136 bp, 120,881 bp, and 89,413 bp.We sequenced the genome of  Roseibium species  are ed genus , 4, 5. WRoseibium sp. strain Sym1 was isolated  .isolated  from a 0isolated  from thestricto) . Brieflystricto) , followeRoseibium sp. Sym1 was revived from a cryostock and inoculated into 25 mL of marine R2A broth. Once high density was achieved, 25 μL of the seed culture was used to inoculate 25 mL of marine R2A broth in triplicate into cell culture flasks for 72 h at 27°C. Inoculum from one flask was used for paired-end sequencing, and inocula from the other two flasks were used for long-read sequencing. Genomic DNA was extracted using the GenElute bacterial genomic DNA kit . A paired-end library was constructed using the Illumina DNA library prep kit and sequenced on the NextSeq 2000 platform, generating an output of 21,815,230 raw short reads (2 × 151 bp). Oxford Nanopore’s genomic DNA by ligation kit was used for long-read library preparation (no size selection); the library was sequenced on the MinION platform using an R9 flow cell chemistry (R9.4.1), followed by high-accuracy base calling using Guppy v5.0.16, which generated an output of 165,651 raw reads with an N50 value of 8,039 bp. Library preparation and sequencing were conducted by SeqCenter (USA). Quality control was conducted using fastp v0.12.4 . UsCP114786, CP114787, CP114788, CP114789, and CP114790. The BioProject accession number is PRJNA884430. The code used to assemble the genome is available at https://github.com/symbiotic-em.The hybrid assembly is available at NCBI GenBank under accession numbers


We recorded a total of 59 macroinvertebrate taxa belonging to 32 families in seven insect orders plus Amphipoda and Gastropoda. Species diversity declined with higher impact. Five environmental factors structured macroinvertebrate community composition in RDA: elevation of sample location, site total nitrogen, dissolved oxygen, electrical conductivity, and water chemistry. We conclude that macroinvertebrate communities are an appropriate and inexpensive tool for monitoring water quality in Mongolia and suggest government action to establish a long-term monitoring program.Mongolia is a country with a quickly growing economy mainly based on mining of gold, copper, coal, and other minerals. Mining, urbanization, and agriculture impact the water quality in the upper Selenga River Basin in northern Mongolia, which is the center of the Mongolian economy. Previous measurements of pollution loads were alarming, but restricted to chemical measurements. Here, for the first time, we combine freshwater biomonitoring and laboratory water quality data across a broad gradient of water quality and land use intensity. We track the effects of different types of pollution on aquatic invertebrates and test their use as bioindicators. We collected water samples, environmental parameters, and macroinvertebrates at 36 sampling sites at the rivers of Tuul, Kharaa, and Orkhon and their tributaries Sugnugur, Boroo, Sharyn Gol, Gatsuurt, and Yeröö. PCA of catchment water quality distinguished three groups of pollutants prevalent at the sites: (1) nutrients, (2) saline components (ClThe online version contains supplementary material available at 10.1007/s10661-023-12022-x. River water quality is under serious global threat from the increasing demands of the world’s growing population, rapid economic development, and the effects of climate change , which are key agricultural, industrial, and mining areas (in particular for gold and copper). This not only increases water consumption, but also increases the chance of water contamination. Although Selenga’s tributaries flow largely through rural areas, there is point and non-point pollution from industrial mining and artisanal gold panning operations, from intensive livestock and scattered crop farming, and from municipal discharges. Treated and untreated sewage is contributing to pollution, which has the potential to harm a large number of people , the capital of Mongolia, with a population of about 1.5 million. Here, the small Selbe River joins into the Tuul. Further downstream the Tuul is heavily polluted by discharges from UB’s central wastewater treatment plant (WWTP), which contain high levels of nutrients, oxygen-depleting substances, and chemicals that can significantly affect water quality  in the laboratory of the first author at the National University of Mongolia using the latest local and international literature and keys  using an aquatic kick net (0.25 × 0.25 m area and of 250 μm mesh size). The kick net is used to penetrate the river bed to a maximum depth of 0.3 m and filter out free-floating organisms attached to the substrate by moving the bottom substrate within an area of approximately 1 mn = 36).For each of the sample sites Figs. ; Table 1vegan , NO3− (mg/L), NO2− (mg/L), PO43− (mg/L), Fe (mg/L), Cu (mg/L), Cr (mg/L)). The formula and its evaluation scheme are given in the Online Resource  for each sampling site, we took eight chemical measurements (DO (dissolved oxygen) (mg/L), NHWe used the Kolmogorov–Smirnov test to validate the normality of the data. Differences between macroinvertebrate community structure and chemical-physical factors at the sampling sites were examined by one-way analysis of variance (ANOVA).4-N (mg/L), total As (µg/L), B (µg/L), Ca2+ (mg/L), total Cr (µg/L), Cl- (mg/L), Co (µg/L), total Cu (µg/L), total Fe (mg/L), PO43- (mg/L), K (mg/L), Mg2+ (mg/L), Mn (mg/L), Mo (µg/L), Na (mg/L), Ni (µg/L), NO3-N (mg/L), NO2 -N (mg/L), Pb (µg/L), SO42- (mg/L), Sr (µg/L), U (µg/L), V (µg/L), Zn (mg/L)).We applied principal component analysis (PCA) to reduce the number of environmental variables and extracted principal components from the main chemical parameters , NHR2 and AIC in parsimonious RDA and calculated the variance inflation factor (VIF) to exclude covariates with VIF > 5.5.Macroinvertebrate communities were assessed by redundancy analysis (RDA) with Hellinger transformation of taxon data .We took a The physicochemical parameters for each of the sampling sites are shown in the Online Resource  = 27.5 p = 0.00001): (A) low impact sites, including our no-impact site T1 and other upstream sites such as K1 and Y1. (B) Sites with higher levels of nitrogen (NH4+), but low metal concentrations, e.g., T3 and K8 downstream of WWTPs in UB and Darkhan, but also many sites along the Kharaa River. (C) Sites in the positive ordination space of the second PCA axis that were affected by higher concentrations of B, Sr, U, Mo, and ions , including urban and mining sites. (D) Sites in the positive ordination space of the first PCA axis that were affected by higher loads of arsenic and heavy metals. This included the mining sites S2, B1, and the arable area Ba1, where we found total metal contents of 75 mg/L, 54 mg/L, and 56 mg/L in the water, respectively, as well as all the downstream rural Orkhon sites affected by the upstream mining in Erdenet and Zaamar, which had total metal contents between 18 to 22 mg/L.A PCA explained 82% of the cumulative variance in the data  and Selbe River Se1 (4.0 mg/L). Urbanization (yellow squares) increases general water pollution as indicated by higher concentrations of chloride and boron. Boron (B) compounds enter wastewater mainly via detergent ingredients, but also with fertilizers and from mining tailings  concentrations in our study ranged from a very low 0.8 mg/L in T1 at the upper Tuul to 17.3 mg/L in Sharyn River S3, with high values in the small tributary Selbe River within UB (Se1: 16.2 mg/L) and in the mining areas of Zaamar downstream of the Tuul .Rural sites  with those of uranium (U) , molybdenum (Mo) , and strontium (Sr) . The highest loads for U (21.1 µg/L) and Mo (9.6 µg/L) were found in the mining area of the Kharaa River upstream of the confluence with the Boroo River (K2), together with the maximum record for Sr (510 µg/L) and a high load of Ca2+ (54.3 mg/L). The highest values for Ca2+ (54.8 mg/L), Cl− (17.3 mg/L), Mg2+ (19.6 mg/L), and Na+ (39.3 mg/L), together with high values for Mo (7.4 mg/L), Sr (576 mg/L), and U (14.8 mg/L), were recorded by S3 at Sharyn River, a site 28 km downstream of the mine at Khuiten-Sharyn (S2). The highest concentrations of Sr (785 mg/L) and SO42− (27.1 mg/L) were recorded at Selbe River (Se1) in UB  and clays and is a determinant of water hardness. In our samples, the concentrations of CaMining activities are responsible for high loads of arsenic and heavy metals in the nearby rivers. Water flow rate is an important parameter and small tributaries can carry higher concentrations. Particularly high levels of arsenic (As) from mining areas were recorded in the Gatsuurt , Kharaa (K2-12.6 µg/L), and Khuiten-Sharyn (S2-10.3 µg/L) streams. S2 had also the highest levels of vanadium (V) (35.6 µg/L), nickel (Ni) (15.2 µg/L), and copper (Cu) (32.1 µg/L). High metal concentrations were also measured at Boroo Mining (B1), where we found high concentrations of iron (Fe 11.6 mg/L) and lead (Pb 9.8 µg/L).In contrast, the Zaamar mining areas on the wide lower reaches of the Tuul River had much lower values, e.g., T7 for As 7.0 µg/L, Cu 9.0 µg/L, Ni 7 µg/L, U 2.7 µg/L, V 16.7 µg/L , Diptera , Ephemeroptera , Hemiptera , Plecoptera , and Trichoptera  were significantly abundant among the sampling sites. Ephemeroptera was also the most abundant order, occurring at all sites and accounting for 59% of total abundance. Among them, Baetis was the most abundant genus, with 37.7% of the total abundance and 65.3% of all individuals of Ephemeroptera. Diptera and Hemiptera were common (13.07% and 15.71%)  in all sampling sites and it was only more abundant in the relatively pristine sites, at Sugnugur (Su1) and Sharyn upstream (S1), both headwaters. Here, Agnetina was a common genus and was recorded in fourteen sampling sites with few individuals.A total of 7368 individuals of macroinvertebrates (59 taxa) were collected from 36 sampling sites in northern Mongolia, belonging into eight orders , 32 families , and 55 genera of insects , we collected 16 taxa. The highest Shannon diversity, 9.76, was calculated for T1, our reference site on the upper Tuul River, representing 15 taxa with a relatively high Shannon evenness of 0.65 Table . This siOnly four taxa were collected at the six sites with the lowest taxon richness, B1, K5, O1, T3, T7, and Se1. Shannon diversity N1 of these sites ranged from 1.40 to 3.31, with B1, a mining site upstream of Boroo, having the lowest values for Shannon diversity and evenness Table .p < 0.05) with temperature (N1 =  − 0.35), TNb , and metal concentrations, viz. Al , Cu , V , and with higher significance (p < 0.01): Cr , Mn , Ni , Pb . However, the correlations with U and Zn were not significant.Species richness and diversity were significantly negatively correlated , decreasing progressively with increasing impact and with the lowest values recorded for the urbanization sites .Fig. R2 = 0.56, F = 9.38, p < 0.001). Shannon diversity increased with elevation and was negatively affected by higher levels of total nitrogen  = 11.13, p < 0.001). Simpson diversity at the sites was determined by elevation and longitude of the sites  = 7.21 p < 0.01). The strong effect of longitude is due to the general flow direction of the Selenga’s tributaries from southeast to northwest; thus, higher longitude is—like altitude—an indicator of upstream sites.We applied multiple regressions to identify the factors that drove macroinvertebrate diversity in our study .For each of the communities at the sample sites, we calculated the biotic index, which measures the sensitivity of aquatic organisms as an indicator of water quality. A lower index number indicates a lower tolerance to pollution and is therefore evidence of a better water quality. The highest index was found at K8, in Kharaa downstream of the Darkhan WWTP, while the lowest was found at the headwaters of Sugnugur, Su1  and altitude , but positively correlated with temperature , pH , EC , chloride , and water chemistry PC2 . A multiple regression with forward selection of variables explained 51% of the variation and showed that the biotic index was driven by Cl− and NH4+ concentrations, while altitude had a negative effect  = 12.37, r2 = 0.51, p < 0.00002, Online Resource Table The index was also negatively correlated with latitude (indicspecies identified six indicator taxa for no-impact sites of the land use grouping: Agrypnia (Phryganeidae), Anagapetus and Glossosoma (Glossosomatidae), and Ironodes and Ecdyonurus (Heptageniidae) were found exclusively at no-impact sites , while 92% of the abundant Apatania (Apataniidae) were recorded there . Three indicator taxa could be assigned to the water quality groups of the PCA: Arcynopteryx (Perlodidae)  and Heptagenia (Heptageniidae)  indicated group A of water quality sites of the PCA, while midges of the family Orthocladiinae (Chironomidae) indicated group C .We searched for bioindicator taxa that should indicate a particular type of land use Table  or one op < 0.05) positively correlated with high concentrations of Mo (R = 0.39), U (R = 0.34), and Zn (R = 0.34). The highly sensitive EPT complex (Ephemeroptera-Plecoptera-Trichoptera) was negatively correlated (p < 0.05) in its abundance with concentrations of Cl− (R =  − 0.40) and TNb (R =  − 0.40). Plecoptera abundance alone was negatively correlated (p < 0.01) with Cr (R =  − 0.47), Cl− (R =  − 0.49), Na + (R =  − 0.50), V , and others, as well as with PC1 . Trichoptera were negatively correlated with Cu  and Ni .We further evaluated the association of specific taxa or taxa groups with water chemistry using Spearman correlation. Odonata were found to be significantly  mac2+, Cl−, Na+, SO42−, and Sr, which are characteristic of mining effluents.Along the RDA1 axis, macroinvertebrate communities are sorted along a gradient of dissolved oxygen (DO) and EC, with the rural sites on the left, with high DO and low EC, followed by urban sites in the middle of the gradient, while mining sites are concentrated on the right, indicating high EC in the water. PC2, the second axis of the PCA of water chemistry, also points in this direction, being strongly correlated with the values of CaF = 1.98, R2adj = 0.13, P < 0.001).In contrast, there is a nitrogen gradient along the RDA2 axis, with the highest values at T3, the central WWTP of UB; Se1, the Selbe River, a small tributary of the Tuul that flows directly through a ger district (yurt settlement) of UB; T5, the mining area in Zaamar where many people work without adequate sanitation; and K8, the WWTP of Darkhan, the second largest city in Mongolia. As UB is located on the upper reaches of the Tuul River at an elevation of 1224 m, the highly concentrated wastewater is slowly degraded as it flows downstream to the confluence of the Orkhon and Selenga Rivers (O4) at an elevation of 608 m. The downstream sampling sites on the Orkhon River are all located in the lower right-hand corner of the figure. The mines on the Gatsuurt River (G1-G3) are also very high, between 1139 and 1020 m. The macroinvertebrate communities of the headwater streams  differ only slightly from those of the mountain streams in the mining areas and are close together in the RDA figure. The slightly irritating position of the no-impact site T1 between the mining sites of the Gatsuurt River is due to its high altitude and the low oxygen content of this quietly flowing river , as confirmed by symmetric Procustes rotation.The RDA scores were significantly correlated with the PCA scores of the sample sites  and other mining by-products , and (D) (heavy) metals.Our results demonstrate that mining, urban development, and agriculture affect water quality and macroinvertebrate communities in different ways. The PCA of the water quality separated a group (A) of unpolluted sites from three other clusters, which were contaminated with various pollutants, namely (B) nutrients, (C) salt components  structured macroinvertebrate communities. Comparable results have been reported from tropical Mexico, where macroinvertebrate assemblages were structured by total phosphorous, water and air temperature, and Hg concentration  was recorded at the agricultural site Ba1, where the total metal centration was 56 mg/L. The stonefly family Perlidae can accumulate metals  had its highest abundance in the mining affected sites S1 and S2, where extra-high concentrations of U, V, Mo, and Al were measured, with total metal contents of 24 mg/L and 75 mg/L, respectively. Similarly, Diptera of the genus Simulium had their peak abundance at metal-impacted Ba1 and S1, and are known to tolerate high total metal contents Below is the link to the electronic supplementary material.


Environmental factors difference is the key factor for the difference in the production, transformation and accumulation of effective components in plants. UPLC-MS/MS and multivariate statistical methods were applied to describe the region difference of amides compounds in Chinese prickly ash peels from different regions and their correlation with climatic factors and soil factors.P < 0.01). Except for hydroxy-γ-sanshool and ZP-amide A, the residual amides contents were significantly positively correlated with organic carbon, available nitrogen, phosphorus and potassium in soil and negatively correlated with soil bulk density. Low temperature, low precipitation and high organic carbon in soil were conducive to amides accumulation.Amides compounds contents were significantly higher in high altitude areas, with obvious altitude change trend. Two ecotypes were classified based on the amides compounds contents, one was the high altitude-cool type from Qinghai, Gansu, Sichuan and western Shaanxi province, and the other one was low altitude-warm type from eastern Shaanxi, Shanxi, Henan, Hebei and Shandong province. Amides compounds content were negatively correlated with annual mean temperature, max temperature of warmest month, mean temperature of wettest quarter and mean temperature of warmest quarter (This study aided in site specific exploration of high amides contents yielding samples, enriched the environment factors effects on amides compounds, and provided scientific foundation for the improvement of Chinese prickly ash peels quality and the location of high-quality production areas.The online version contains supplementary material available at 10.1186/s12870-023-04328-2. Chinese prickly ash is a characteristic spice in China, mainly produced in Gansu, Sichuan, Shaanxi, Shanxi, Henan and other provinces , 2. PungThe biosynthesis and accumulation of effective components in plants are affected by multiple factors of geographical environment  , 11. TheChinese prickly ash germplasm resources are abundant in China, most of which are wild and semi-wild and concentratedly distributed in the semi-humid and semi-arid areas of the Loess Plateau and the dry and hot valley zone in the Qinba Mountains , 21. UndIn the present study, 26 wild Chinese prickly ash peels samples and 26 corresponding soil factors were collected from different regions in China. The combinations of UPLC-MS/MS and multivariate statistical approach were applied to describe the regions variation of amides in wild Chinese prickly ash peels from different regions, and to interpret the correlation between the amides compounds and ecological factors (climatic factors and soil factors). These results revealed the regional differences of amides in Chinese prickly ash peels from natural distribution area, and elucidated the key environmental factors affecting the amides accumulation, which provided a scientific basis for the protection and utilization of excellent wild Chinese prickly ash germplasm resources, the quality improvement and the selection of high-quality producing areas.Wild Chinese prickly ash fruit samples were collected fron its natural habitat in China. The mature fruits and their rhizosphere soil were collected by field investigation from 26 sites covering 8 provinces of China at different altitudes  (Each sample was collected once a year) from July to September of 2019 and 2020. The detailed sampling information was shown in Fig. The preparation of dried Chinese prickly ash peels was based on methods published in the literature as follows : (1) cruThe amides compounds extracts were analyzed using an UPLC-ESI-MS/MS system  by MetWare . The UPLAnalyst 1.6.3 software was applied to analyze the amides data in Chinese prickly ash peels from different regions. The peak area of amide metabolites was corrected by MultiQuant 3.02 , and the corresponding relative content of amides was expressed by chromatographic peak area.https://www.resdc.cn/). 19 climate environment variables were obtained from the WorldClim database (version 2.1), with spatial resolution of 30s (about 1km2). ArcGIS 10.7 software was applied to extract climate data corresponding to the latitude and longitude data of 26 sampling sites. All the climate factors were listed in Table The annual mean temperature, annual mean precipitation, annual mean sunshine hours and other meteorological data of sampling points were obtained from the Resource and Environmental Science and Data Center of the Chinese Academy of Sciences (Soil bulk density and gravel contents were measured by ring knife method. Semi-micro Kjeldahl method GB7173-87) was carried out to determine total nitrogen content, sodium hydroxide molten molybdenum antimony anti-colorimetric method (GB8937-88) was used to measure total phosphorus content, sodium hydroxide alkali fusion atomic absorption spectrometry was used for determination of total potassium content (GB9836-88), and soil organic carbon content was determined by combustion oxidation-titration (HJ 658–2013) [3-87 was Chemometric analyses such as hierarchical cluster analysis (HCA), principal component analysis (PCA), orthogonal partial least squares-discriminant analysis (OPLS-DA), correlation analysis (CA), path analysis (PA) and structural equation model (SEM), were employed to systematically analyze the geographical variation of amides contents in Chinese prickly ash peels from different regions and the relationship between environmental factors and amides contents. Among them, HCA, PCA, OPLS-DA and CA were generated using the Origin software for statistical and computing , and PA and SEM were performed using SPSS 24.0 for Windows .Significant differences in the total amide contents in Chinese prickly ash peels were obviously observed Fig. . The amiUPLC-MS/MS was applied to determine amide compounds in Chinese prickly ash peels from different rehions, and 13 amide compounds were identified, including γ-sanshool, ZP-amide N, dihydrosanshool, tetrahydrosanshool, bungeanool, hydroxy-β-sanshool, hydroxy-α-sanshool, hydroxy-γ-sanshool, dehydroxy-γ-sanshool, ZP-amide C, ZP-amide J, ZP-amide M, ZP-amide A. Among them, ZP-amide C was not detected in S12, S14, S15, S16, S17, S21 and S22, and dehydroxy-γ-sanshool and ZP-amide J were not detected in S18 and S20 (Table The main amides in Chinese prickly ash peels were hydroxy-α-sanshool (24.38–66.24 mg/g), hydroxy-β-sanshool (8.34–12.39 mg/g) and hydroxy-γ-sanshool (3.27–15.45 mg/g), with the average contents of hydroxy-α-sanshool, hydroxy-β-sanshool and hydroxy-γ-sanshool were 44.14 mg/g, 10.77 mg/g and 7.18 mg/g, respectively. The contents of hydroxy-α-sanshool, hydroxy-β-sanshool and hydroxy-γ-sanshool accounted for 74.06% of the total amount of amides. The hydroxy-α-sanshool content was the highest, which was the major amide compound to determine the numb taste of Chinese prickly ash peels. Among them, the hydroxy-α-sanshool contents of S8, S9, S11, S2 and S3 were higher than 63.00 mg/g, and hydroxy-α-sanshool contents of S20 and S24 were the lowest, with the content of 24.97 ± 0.31 mg/g and 24.38 ± 0.49 mg/g, respectively. The hydroxy-β-sanshool contents of S1, S6 and S11 were the highest, which were 12.39 ± 0.31,12.62 ± 0.35 and 11.82 ± 0.33 mg/g, respectively, whereas that of S7, S18 and S24 were the lowest. The hydroxy-γ-sanshool contents in S22, S23 and S24 were the highest, which were 15.45 ± 0.36,12.41 ± 0.16 and 12.09 ± 0.25 mg/g, respectively. The hydroxy-γ-sanshool contents in S1, S2 and S3 were 3.27 ± 0.22,4.20 ± 0.16 and 3.44 ± 0.09 mg / g, respectively. The γ-sanshool content was 1.25 to 7.68 mg/g, with the average content of 2.99 mg/g. ZP-amide N content was 0.05 to 6.25 mg/g, with an average content of 1.87 mg/g. The content of dihydrosanshool was 0.34 to 4.43 mg/g, with an average content of 2.67 mg/g. Tetrahydrosanshool content was 0.48 to15.35 mg/g, and the average content was 6.30 mg/g. Bungeanool content was 0.38 to 5.23 mg/g, and the average content was 2.84 mg/g. Dehydroxy-γ-sanshool content was 0.84 to 7.50 mg/g, and the average content was 2.52 mg/g. ZP-amide C content was 2.72–8.96 mg/g, and the average content was 4.34 mg/g. ZP-amide J ranged from 1.12 to 2.56 mg / g, and the average content was 1.76 mg/g. The average content of ZP-amide M was 0.97 mg/g, ranging from 0.05 to 2.71 mg /g. ZP-amide A ranged from 0.11 to 1.56 mg/g, with an average content of 0.56 mg/g.The variation coefficient of 13 amides ranged from 12.07 to 86.88%, with the average variation coefficient of 53.91%. The variation coefficients of ZP-amide N, tetrahydrosanshool, ZP-amide C, ZP-amide M and ZP-amide A were higher than 64%, which indicated that the differences of amide compounds in Chinese prickly ash peels from different regions were larger. The variation range of hydroxy-β-sanshool was 12.07%, which demonstrated that the hydroxy-β-sanshool content in Chinese prickly ash peels from different regions was stable.To further elucidate the regional difference of amide compounds contents in Chinese prickly ash peels from different regions, chemometric analyses such as hierarchical cluster analysis (HCA), principal component analysis (PCA), and orthogonal partial least squares-discriminant analysis (OPLS-DA) were applied to illustrate the region variation of amides and screen out the key amide substances that distinguish and evaluate Chinese prickly ash peels from different regions.PCA and HCA were performed to clarify the differences and similarities of amide compounds in Chinese prickly ash peels from different region, and 13 kinds of amides were used as variables. The eigenvalues of the first three principal components were greater than 1, and the cumulative variance contribution rate reached 74.720% , indicating that 13 amides could distinguish Chinese prickly ash peels from different regions. The PCA results were shown in Fig. The furthest neighoor’s method was put in application to sort samples into groups, and the pearson correlation distance was selected as the similarity measure standard, and the HCA results were described in Fig. Although the PCA method could effectively extract the main information, it is not sensitive to variables with low correlation. OPLS-DA analysis is a multivariate statistical analysis method for supervised pattern recognition, which combined orthogonal signal correction and partial least squares-discriminant analysis to maximize the distinction between groups and could screen out the key differential compounds through variable importance projection (VIP).The OPLS-DA score scatter diagram of Chinese prickly ash peels from different regions displayed that 26 samples was distinguished at the first principal component (42.8%), and could be divided into two groups  of the OPLS-DA model, 7 amide compounds with greater contribution to the differentiation were selected Fig. C. It canγSS, YAN, YDDBG, YTDBG, YBG and YHαSS were negatively correlated with bio1, bio4 , bio5 (max temperature of warmest month), bio7 , bio8 (mean temperature of wettest quarter) and bio10 (mean temperature of warmest quarter) (P < 0.01), and positively correlated with bio3  (P < 0.01), which suggested that high temperature and temperature fluctuation were not conducive to amides formation. YγSS, YAN, YDDBG, YBG and YHαSS exhibit a negative correlation with annual sunshine duration (P < 0.05). Except for YHβSS, the remaining amides compounds were positively correlated with bio12 . YHγSS was positively correlated with bio1, bio4, bio5, bio8 and bio10, and negatively correlated with bio3, indicating that high temperature in the growing season was conducive to the YHγSS biosynthesis. YAM and YAA displayed a positive correlation with bio3 (P < 0.05), and a negative correlation with bio4, bio5 and bio10 (P < 0.05). YAA was positively correlated with bio12, bio16 and bio18 (P < 0.05), indicating that low temperature and sufficient precipitation were conducive to the YAA accumulation. Most of the amides were positively linked to wind speed. In short, the low temperature and less precipitation from coloring period to mature period were conducive to amides compounds accumulation, and could help to improve the Chinese prickly ash peels quality.The production and accumulation of amides contents in Chinese prickly ash peels from different regions were regulated by climate factors. The correlation analysis results exhibited that there were different degrees of correlation between amides and climatic factors, and the results were shown in Fig. In addition, the correlation between climatic factors and amides contents suggested that the climatic conditions in Hanyuan, Wudu, Qin’an, Wenxian and Fengxian were conducive to the amides accumulatio. In order to produce Chinese prickly ash peels with excellent quality, the semi-humid and semi-arid areas in the Qinba Mountains and the hilly and gully areas of the Loess Plateau  should be selected as high-quality producing areas for large scale cultivation of Chinese prickly ash.γSS, YAN, YDDBG, YTDBG, YBG and YHαSS were positively correlated with XCEC, XN, XK, XP and XSOC (P < 0.01), and negatively correlated with XBD (P < 0.01) , and YAA was negatively correlated with XPH, XBD and XTH (P < 0.05). In summary, it demonstrated that soil total N, P, K, soil organic carbon, cation exchange capacity and soil bulk density had a great effect on amides accumulation. The soil PH value of sampling sites had no significant effect on amides accumulation.Different soil physicochemical properties also had significant effect on the accumulation of amides compounds in Chinese prickly ash peels. Y01) Fig. . YAM andTo gain insight into the response characteristics between environmental factors and amide compounds content, path analysis (PA) was performed to calculate the direct and indirect effects of environmental factors on amide content. Taking 13 kinds of amides as independent variables and environmental factors as dependent variables, the direct and indirect path coefficients of dominant environmental factors were calculated by stepwise regression analysis.TDBG, XP and bio 7 on YHαSS, and XN on YHγSS, indicating that the environmental factors had direct and decisive effects on the biosynthesis and accumulation of amide compounds.The PA results of Table  in this N had a positive direct effect (0.659) on YHαSS, and a negative direct effect (-0.54) YHγSS. XMW had a positive direct effect (0.173) on YγSS, but with the negative indirect effect (-0.182) and the positive correlation coefficient (0.355), suggesting that the indirect effect XMW on YγSS through bio5 and bio4 was the contributory cause of relevance. Bio4 had both negative direct and indirect effects on YγSS and YTDBG, but with a positive indirect effect (0.232) on YBG. Bio5 had both negative direct and indirect effects on YγSS and YAN (P < 0.05), and the direct effects were higher than the indirect effect, which demonstrated that bio5 played a direct and decisive role in YγSS and YAN accumulation. Bio17 had negative direct effects on YHβSS and YHαSS (P < 0.05), but to YHαSS, displayed a positive indirect effect. XSOC was the key environment factor for the contents of YAN and YHαSS (P < 0.05). XK was the key factor for YDHγSS, with a positive direct effect (0.451), and XCEC was the key factor for YAA, and had a positive direct effect (0.651). In a word, path analysis illustrated the relative contribution rate of various environmental factors to amides, which made the multivariate statistical analysis more rational.XThe structural equation model (SEM) is a statistical method for analyzing the relationship between variables based on the correlation coefficient or covariance matrix. The relationship between total amides contents, main amides components and environmental factors was expounded by SEM, and the SEM model fitting analysis with CMIN/DF < 1, RMSEA < 0.08 and AGFI > 0.9 was selected, which suggested that prediction results of the model were excellent.HαSS, YγSS, YTDBG and YHγSS had significant direct effects on total amide content, and the path coefficients were 0.96 (P < 0.01), 0.90 (P < 0.01), 0.93 (P < 0.01) and − 0.62 (P < 0.01), respectively . XTH, bio10 and bio4 had significant negative effects on total amide content, and the path coefficients were − 0.83, -0.69 and 0.99, respectively; and the indirect path coefficients through YHαSS were 0.93 (0.97 × 0.96), -0.16 (-0.12 × 0.96) and 0.16 (0.12 × 0.96), respectively  and 0.78 (P < 0.01), respectively, and their indirect effects through YγSS on total amide content were 0.09 (0.11 × 0.90) and − 0.55 (-0.61 × 0.90), respectively. The indirect effect of bio10 through YγSS on total amide content was − 0.67 (-0.74 × 0.90) , -0.54 (-0.58 × 0.93) and − 0.51 (-0.55 × 0.93), respectively  and − 0.38 (0.62×-0.62). XN and XP exhibited significant direct effects on total amides content, with the direct path coefficients of 0.70 (P < 0.01) and 0.57 (P < 0.01), respectively  were higher. To meet the surging demand, the above regions could be selected for large scale cultivation of Chinese prickly ash, which was consistent with the actual producing area of Chinese prickly ash recognized as a good variety in China .Climate factors could play an important role in regulating the synthesis and accumulation of the effective components in plants –29. The In our research, γ-sanshool, ZP-amide N, dihydrosanshool, tetrahydrosanshool, bungeanool, hydroxy-α-sanshool and hydroxy-γ-sanshool were negatively correlated with annual temperature, max temperature of warmest month, mean temperature of wettest quarter and mean temperature of warmest quarter. The main amides in Chinese prickly ash peels  were negatively correlated with the precipitation of wettest month and precipitation of warmest quarter. The study area belongs to the hydrothermal synchronization, indicating that high temperature was not conducive to amides formation in the development stage and mature stage. High temperature is the main meteorological disaster from coloring period to mature period. Excessive high temperature would reduce the activity of enzymes, affect the conversion of substances, and be not conducive to the conversion of sugar to downstream products, which was not conducive to the amides formation and accumulation. The amides were easy to be degraded in high humidity environment with air humidity over 80%. In short, low average temperature and less precipitation from coloring period to mature period were conducive to amides accumulation.+ or other cations exchange out at any time, supply pepper Na+, K+, Ca2+, Mg2+ and some trace nutrients [Soil was the basis for plant nutrient conservation and transformation, and plant growth and development and the effective component contents in plants were determined by soil fertility , 32. Theutrients , 39. Potutrients , 41. Nitutrients . Nitrogeutrients , 44. PhoAlkylamides are considered a combination of unsaturated fatty acids, valine, or phenylalanine. Alkylamide compounds have been proven to be the key components in Chinese prickly ash peels, but their biosynthesis pathways remain largely unknown. To fully elucidate the mechanisms by differetnt ecological factors induce amides synthesis and accumulation, the expression profiles of Chinese prickly ash peels cultured with different topographical conditions require investigation. Based on homologue searching, transcriptome analysis and ectopic expression, we would identified the candidate genes that may have contributed to amides synthesis in Chinese prickly ash peels under different ecological factors in the future experiments. Future experiments could cmobine with the present high-quality reference genome sequence leverage these findings to increase peels quality in Chinese prickly ash. Overall, this study provides valuable new insights into the amides accumulation underlying the responses of Chinese prickly ash peels to ecological factors.The total amides compounds contents in wild Chinese prickly ash peels from different regions were significantly different, with obvious altitude change trend. The contents of hydroxy-α-sanshool, γ-sanshool, ZP-amide N, hydroxy-γ-sanshool, dihydrosanshool, tetrahydrosanshool and bungeanool in peels from high altitude area were significantly higher than those in low altitude areas, and the numb-taste intensity was higher. The annual mean temperature, max temperature of warmest month, mean temperature of wettest quarter, mean temperature of warmest quarter, soil organic carbon, available nitrogen, phosphorus, and potassium were the main environmental factors affecting amides compounds accumulation. Different effects of climate and soil factors on amides contents were applied to optimized factor, select suitable agroclimatic zones for cultivation of selective varieties with high content of amide compounds. This aided in sites specific exploration of high amides yielding samples, and promoted the agriculture practices of Chinese prickly ash in regions with similar phytogeographical location.Below is the link to the electronic supplementary material.Supplementary Material 1


Introduction: The product labels of veterinary disinfectants specify their expiration dates to prevent the use of outdated products, as these may result in disinfection and biosecurity failures during outbreak situations. However, a clear standard for the storage conditions of diluted disinfectant solutions has not yet been established, and the effects of storage conditions have scarcely been investigated. To fill this research gap, our study examined the stability of the active ingredients of diluted veterinary disinfectants based on their change in concentrations when stored at various temperatures for various time periods.Methods: Twenty veterinary disinfectants effective against either foot-and-mouth disease or avian influenza viruses were selected. The disinfectants were diluted to effective concentrations following the manufacturer’s instructions. Using selective analytical techniques, the concentrations of the active ingredients of the samples that had been stored for varying intervals at different temperatures  were determined. These samples included soaps and detergents, acids, oxidizing agents, aldehydes, and copper compounds. The active ingredient concentrations of two of the samples were determined following freezing/thawing cycle, to establish their stability when exposed to simulated winter conditions.Results: Our results showed that most of the active ingredients had concentrations of 90% or greater of their initial concentrations, indicating ≥90% stability over a 21-day period under the experimental storage conditions. However, there were some exceptions. Glutaraldehyde, formaldehyde, and malic acid are over 90% stable at ≤ 30°C for 21 days, but their concentrations decreased to below 90% of their initial concentrations at 45°C, indicating a decline in stability when stored at 45°C for 21 days. The concentrations of potassium peroxymonosulfate and peracetic acid rapidly declined with increasing time and temperature to less than 90% of their initial concentrations.Discussion: Based on our findings, we propose that diluted disinfectant solutions should preferably be prepared daily. However, if the daily preparation of a diluted disinfectant solution is not feasible, then our results can be used as a reference, providing basic scientific data on the chemical stability of diluted disinfectant solutions commonly used in the veterinary field, thus indicating suitable storage conditions. The active ingredients in a disinfect are the chemicals responsible for inhibiting the growth of microorganisms. The contents of these ingredients are listed as percentages or concentrations on the product label, along with their respective expiration dates. The expiry period is based on the stability of the active ingredients, known to decline over time owing to several chemical processes triggered by various conditions.In South Korea, the Animal and Plant Quarantine Agency (APQA) performs quality control of commercial veterinary disinfectants, both through an official approval step and an annual post-market surveillance study, evaluating their efficacy against the targeted microorganisms and the contents of the active ingredients in the final product . CurrentDisinfectants are classically grouped into soaps and detergents, alkalis, acids, chlorine and chlorine compounds, oxidizing agents, aldehydes, phenol compounds, quaternary ammonium compounds (QACs), and alcohols . The WorEscherichia coli and Staphylococcus aureus at concentrations ranging from 10 to 0.002 g/L and at contact times of 30, 60 and 90 min. Other authors have summarized the studies on the common chemical disinfectants and their virucidal efficacies under various application conditions. Numerous studies have investigated the efficacy of active ingredients or disinfectants against microorganisms; at specific concentrations, for given time intervals and/or various temperatures . Phenol However, research on the stability of active ingredients, based on concentration changes in diluted veterinary disinfectant solutions under certain storage conditions, is scarce and there is a lack of scientific evidence regarding the relationship between the actual concentration of active ingredients in diluted disinfectant solutions and their efficacies. Moreover, as no clear standard has yet been established for the storage conditions of diluted disinfectant solutions, daily replacement and preparation of fresh dilutions are recommended in most disinfection protocols to ensure optimal efficacy . In infeIn this study we attempted to elucidate how the concentrations of the active ingredients of diluted disinfectant solutions and therefore their stabilities change under various storage conditions. The study included the assessment of the stability of diluted disinfectant solutions when exposed to a freezing/thawing cycle, to investigate the stability of diluted disinfectant solutions during January, known as the coldest month in South Korea with average temperatures reaching −6.9°C to 3.6°C . In the Twenty veterinary disinfectants, approved by the APQA as effective against either foot-and-mouth disease virus (FMDV) or avian influenza virus (AIV), were selected and classified according to their major active ingredients. The categories of active ingredients in disinfectants are detergents, acids, oxidizing agents, aldehydes, and copper compounds. A summary of the disinfectants and the classification of chemical disinfectants is presented in The stability of the active ingredients in the 20 diluted disinfectant solutions was studied at four temperatures  and at different time intervals . The active ingredient of each sample was assessed immediately after preparation (Day 0), prior to storage at the set temperature, and again after removal from storage at the set time intervals, using selective analytical techniques. Additionally, two diluted disinfectant solutions were assayed for changes in the content of active ingredients during a freezing/thawing cycle, i.e., the solutions were frozen after testing on the day of dilution (Day 0), thawed on the next day (Day 1), tested, and frozen again. The active ingredient of a sample was considered stable if it was within 90%–120% of its initial concentration.2O), and formaldehyde  were used for high-performance liquid chromatography (HPLC) analysis. A phosphorus standard solution  was purchased from Sigma–Aldrich  for inductively coupled plasma optical emission spectroscopy (ICP-OES). HPLC-grade water was obtained from Honeywell Burdick and Jackson . For the titration method validation, potassium peroxymonosulfate , hydrogen peroxide , and GLT  were purchased from Sigma–Aldrich, and other active ingredients were obtained from the manufacturers. The standard solutions of quaternary ammonium compounds (QACs), sodium hypochlorite , and copper sulfate pentahydrate  were provided by the manufacturers. The QACs of didecyl dimethyl ammonium chloride (DDAC), alkyl dimethyl benzyl ammonium chloride (ADBAC), and coco dimethyl benzyl ammonium chloride (CDBAC) were of 81%, 50.58%, and 50.12% purity, respectively. All other reagents were of analytical grade.U.S. Pharmacopeia (USP) reference standards for citric acid , malic acid , acetic acid , glutaraldehyde , according to the previously validated and/or slightly modified APQA procedures  was determined for CuSO4.5H2O content analysis , Hocedures . Supplemocedures ; ADBAC wocedures ; and CDBocedures . The conn method . Availabn method , and avan method . The conanalysis . Each ex2PO4) adjusted to pH 2.0 with phosphoric acid; flow rate of 1.0 mL/min; column compartment temperature of 35°C; injection volume of 10 μL; and ultraviolet (UV) detection at 210 nm. The analytical chromatographic separations of GLT and FAL were performed after derivatization with 2,4-dinitrophenylhydrazine (DNPH), on a Zorbax SB-Aq column  before injection. An HPLC system  equipped with a pump (Model G7111 A), autosampler (Model G7129 A), sample thermostat (Model G4761 A), column oven (Model G7116 A), and a diode array detector  was used to analyze CA, MA, AA, GLT, and FAL. Chemstation Software (Version Rev C.01.10 (236)) was used for data processing and evaluation. The analytical chromatographic separations of CA, MA, and AA were performed on a Zorbax SB-Aq column (4.6 The phosphoric acid (PA) content was determined by quantifying the phosphorus in the samples, using an ICP-OES . Phosphorus was determined in the axial configuration, and argon (99.99 v/v%) was used for plasma generation at a flow rate of 1.0 mL/min. The spectral wavelength selected for phosphorus analysis was 213.617 nm. The phosphorus concentration was obtained using a calibration curve. Each experiment was performed in triplicate, and the average values were used for analysis. The results were expressed as a percentage of the results obtained on Day 0 (baseline).To measure the concentration of peracetic acid (PAA), the chronoamperometry test Kemio™  with corresponding sensors for PAA was used. Each experiment was performed in triplicate, and the average values were used for analysis. The results were expressed as a percentage of the results obtained on Day 0 (baseline).Data analyses were performed using Microsoft Excel 2019. All data were expressed as the mean, and significant differences were calculated using an ANOVA. Linear regression analyses were performed on Excel, and the 95% confidence intervals (CIs) were calculated using a reported formula . The act2O2, and NaOCl; aldehydes including GLT and FAL; and CuSO4.5H2O.A total of 14 active ingredients in 20 diluted disinfectant solutions were analyzed for content stability in this study: QACs including DDAC, ADBAC, and CDBAC; acids including CA, MA, PA, AA, and PAA; oxidizing agents including PPMS, HThe analytical methods were validated to demonstrate their suitability for the intended purposes.R2) were ≥0.9998 in all cases. The standard concentration ranges were 10–100 mg for DDAC, 1–10 mg for ADBAC, 5–50 mg for CDBAC, 10–80 mg for GLT, 2–30 mg for PPMS, 5–75 mg for H2O2, 5.5–60 mg for NaOCl, and 5–40 mg for CuSO4.5H2O. Precision was expressed as a percentage relative standard deviation (RSD%), and the obtained RSD% values were: ≤ 1% for DDAC, ≤ 0.5% for ADBAC, ≤ 1.56% for CDBAC, ≤ 0.5% for GLT, < 1% for PPMS, ≤ 1.5% for H2O2, ≤ 1.2% for NaOCl, and ≤ 0.8% for CuSO4.5H2O. These results represent both intra-day and inter-day performances, indicating an acceptable degree of precision for all the titration methods.For the titration method, the titrant volume at the end-point increased linearly with increasing concentrations for each active ingredient. Five tests were conducted for each titration assay, over a standard concentration range covering the concentrations of the compounds in the samples; the regression coefficients (n = 10) of 0.02 μg/mL; the RSD% was 4.72%. The mean recoveries (±SD) and RSD% of the standard solutions were: 94.80% ± 10.31% and 10.87% for 0.1 μg/mL, 106.70% ± 3.56% and 3.33% for 1 μg/mL, and 104.29% ± 0.71% and 0.68% for 5 μg/mL. The LOD and LOQ of the phosphorus content were 0.0037 and 0.0110 μg/mL, respectively.The ICP-OES method, employed for determining the phosphorus content in the PA of the samples, was validated for linearity, LOD, LOQ, precision, and accuracy using standard PA solutions. A linear relationship was obtained over the five-point standard concentration range (0.1–10 μg/mL) with a regression equation of y = 38534x + 387.7 and, a regression coefficient of 0.9998. Intra-assay repeatability was assessed in one laboratory by a single operator, by testing identical samples  were determined using HPLC, whereas the content of the inorganic acid (PA) was determined using ICP-OES. CA was present in the diluted disinfectant solutions of disinfectants B, D, E, G, H, I, and J at concentrations of 0.08%–0.33%. The diluted disinfectant solution of disinfectant J also contained AA at a concentration of 0.040% . PA was 2O2 was present in the diluted disinfectant solutions of disinfectants O, P, and Q at concentrations of 0.03%–0.05%  at 45°C from Day 10 onwards in the samples of disinfectants O and P. Nevertheless, the concentrations were above 90% of the initial concentrations at the end of the experimental storage period  at 45°C during the experimental storage period. GLT exhibited a 51, 10, and 17% content reduction in the diluted disinfectants C, F, and R solutions after 21 days  at 45°C during the experimental storage period, showing a 12% reduction in MA concentration. The lower 95% CI displayed below 90% of the initial concentration on Day 7 at 45°C  under refrigeration storage (6°C) than at elevated temperatures (15°C and 30°C). The effects of temperature on the decomposition of disinfectant solutions vary; however, the concentration of active species in AA and PA solutions typically decreases, and their critical temperatures are 230°C and 450°C, respectively  under refrigerated storage (6°C) than at elevated temperatures (15°C and 30°C). GLT is an active ingredient affected by temperature  for 60 days  and thatperature . Previouperature . Kim et perature . FAL decperature . Liu et 2O2 and PAA as well as PPMS disinfectants should be used within a short period after dilution. In addition, heating as a means to prevent freezing of the disinfectants in winter should be avoided, as heat reduces the PAA and PPMS contents.PPMS and PAA, are used as oxidizing agents. In this study, the concentration of PAA and PPMS decreased over time with increasing temperature. A stability of 90% was shown at 4°C but not under other conditions. The reactive species have to remain stable during storage to maintain their disinfection properties . If the


The protein was precipitated with acetonitrile, the analytes were eluted with acetonitrile and a 0.1% formic acid solution in a gradient program, and lacosamide, ODL, and lamotrigine  were successfully separated by chromatography. The findings of the biological analysis revealed that the lower limit of quantification (LLOQ) for lacosamide in samples was 2 ng/mL and the linearity ranged from 2 to 10000 ng/mL. The LLOQ for ODL was 1 ng/mL, while the linearity range for this substance was 1–1,000 ng/mL. In rat liver microsomes (RLM), the LLOQ of ODL was 80 ng/mL and the linear range was 80–40000 ng/mL. The selectivity, stability, matrix effect and recovery rate were all satisfied with the need of quantitative analysis of samples. Then, the UPLC-MS/MS assay was employed successfully on the interactions of lacosamide and nisoldipine in vivo and in vitro. The half-maximal inhibitory concentration (IC50) was 3.412 μM in RLM, where nisoldipine inhibited the metabolism of lacosamide with a mixture of inhibition mechanism. In rat pharmacokinetic experiments, it was found that nisoldipine could significantly change the pharmacokinetic characteristics of lacosamide, including AUC(0-t), AUC(0-∞), Tmax, CLz/F and Cmax, but had no significant effect on ODL. In summary, the UPLC-MS/MS method could accurately and sensitively quantify lacosamide and ODL, and could be used for the interaction between nisoldipine and lacosamide in vivo and in vitro.Lacosamide, a third-generation novel antiepileptic drug, was first approved in 2008 as an adjunct to partial seizures. In 2014, the U.S. Food and Drug Administration (FDA) approved it as a single agent for partial seizures. Since epilepsy is a chronic condition, most patients need long-term antiepileptic medicinal products, so it is even more important to consider the drug-drug interactions (DDIs). For the purpose of this experiment, an ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay with accuracy and simplicity was optimized and fully validated for the simultaneous quantitative determination of lacosamide and O-Desmethyl-lacosamide (ODL), and DDIs between lacosamide and nisoldipine  Approximately 70 million individuals globally are suffering from epilepsy, among the most prevalent brain disorders. It is distinguished by a propensity to repeatedly cause spontaneous seizures, which have a variety of neurobiological, cognitive, and psychosocial effects . The firLacosamide , as a thAntiepileptic drugs often require other drugs in combination with multiple therapies to control the occurrence of seizures and thus promote the development of drug-drug interactions (DDIs). Therefore, their co-administration with lacosamide may affect their pharmacokinetic and pharmacological effects. In a clinical study, when lacosamide was used in combination with an enzyme inducer of anticonvulsants such as carbamazepine, phenobarbital, or phenytoin, the concentration of lacosamide was lower than when used alone . LacosamIn addition, as global life expectancy increases, the number of elderly epilepsy patients is increasing. Elderly patients with epilepsy are at great risk of cardiovascular disease, and many epilepsy patients with cardiovascular and cerebrovascular diseases in the treatment of the primary disease at the same time to take cardiovascular drugs to control the disease . Due to in vitro results suggested that nisoldipine had a significant inhibitory effect on lacosamide in RLM, we performed in vivo drug interaction experiments in the presence or absence of nisoldipine in rats, and evaluated the changes of pharmacokinetic parameters of lacosamide and ODL.However, to the best of our knowledge, only one analytical method for the simultaneous measurement of lacosamide and its metabolite ODL in biological matrices by LC-MS/MS has been reported. In this investigation, the LC-MS/MS method for the measurement of lacosamide and ODL had long analysis run time (6.5 min) and low sensitivity (0.95 μg/mL) . Thus, iLacosamide , lamotrigine , and ODL, were supplied from Beijing Sunflower Technology Development Co., Ltd . Nisoldipine , Lercanidipine , Nitrendipine , Nimodipine , Lacidipine , Felodipine  and Nicardipine  were provided by Shanghai Canspec Scientific Instruments Co., Ltd. . Acetonitrile (HPLC grade) and methanol (HPLC grade), were produced by Merck Company . Ultra-pure water was prepared by a Milli-Q water purification system manufactured by Milli-Q . All other chemicals and biologicals were of analytical grade or above.A Waters UPLC-MS/MS System comprised of a Waters Xevo TQ-S triple quadrupole tandem mass spectrometer  and a Waters Acquity UPLC I-Class system  was used for chromatographic analysis using an Acquity BEH C18 chromatography column  at a flow rate of 0.40 mL/min. In addition, other conditions were set as follows: injection volume of 6.0 μL, autosampler temperature of 10°C, column temperature of 40°C. 0.1% formic acid in water (solution A) and acetonitrile (solution B) was used to form the mobile phase with a gradient elution as below: 0–0.5 min, 90% A; 0.5–1.0 min 90%–10% A; 1.0–1.4 min, 10% A; and 1.4–1.5 min, 10%–90% A. 90% A was then maintained at 1.5–2.0 min to reach equilibrium. The entire run time was 2.0 min.A Waters Xevo TQ-S triple quadrupole tandem mass spectrometer was combined with electrospray ionization (ESI) in positive ion mode for mass spectrometry. Measurements were conducted by multiple reaction monitoring (MRM). The quantitative ion pairs and related parameters of lacosamide, its metabolite ODL, and IS are listed in g for 1 h. Finally, the supernatant was discarded and the microsomal microspheres were frozen in a 0.01 mM PBS suspension and kept at −80°C. The Bradford Protein Assay Kit  was used to measure the protein concentration (Six blank rat livers were weighed and homogenized with 0.01 mM cold PBS buffer containing 0.25 mM sucrose solution (stored at 4°C). For 15 min, the homogenate was centrifuged at 11,000 rpm. After that, discard the precipitation, and the supernatant was transferred to a fresh tube and centrifuged once again for 15 min at 11,000 rpm. Supernatant was then spun at 100,000 ×ntration .A certain amount of lacosamide was accurately weighed, and diluent (methanol) was added into the EP tube to dissolve. After shaking, 1.00 mg/mL lacosamide reserve solution was obtained. A series of standard working solutions with concentration gradients of 20, 100, 500, 1,000, 5,000, 10,000, 50,000 and 100,000 ng/mL were obtained by dilution with methanol. According to the same method, ODL were prepared and a series of concentration gradients were obtained: 10, 20, 50, 100, 500, 1,000, 5,000 and 10,000 ng/mL. From the prepared standard working solution, 10 μL lacosamide and 10 μL ODL were added to 80 μL blank matrix to prepare calibration standards with concentrations of 2, 10, 50, 100, 500, 1,000, 5,000 and 10,000 ng/mL for lacosamide, and 1, 2, 5, 10, 50, 100, 500 and 1,000 ng/mL for ODL, and these standard curves were determined. Lower limit of quantification (LLOQ) and three Quality Control (QC) samples were obtained by the same method. The LLOQ of lacosamide and ODL were 2 ng/mL and 1 ng/mL, respectively, and the three QC samples of lacosamide and ODL were 5, 4,000 and 8,000 ng/mL, and 2, 400 and 800 ng/mL, respectively. All chemicals and solutions were stored at - 80°C for further use.Calibration curves (80–40000 ng/mL) in RLM were established by preparing calibration products according to the above method. The three QC concentrations were 200, 16,000 and 32,000 ng/mL, respectively, and the LLOQ was 80 ng/mL.Protein precipitation was used to remove proteins from plasma and extract the test materials. 100 μL plasma sample was mixed with 10 μL IS working solution (1 μg/mL) and 300 μL acetonitrile, and centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatant was obtained and aspirated into a sample bottle. Finally, 6.0 μL of sample was injected for analysis.The parameters of this test, which was conducted according to with the FDA bioassay, included selectivity, calibration curve, LLOQ, accuracy and precision, matrix effect, recovery, and stability .Different batches of matrix-match samples were treated to examine the selectivity of the UPLC-MS/MS method. Analysis of blank matrix samples , and real sample is done to determine whether endogenous chemicals interfere with the retentions of the analytes and IS.The concentrations of lacosamide and ODL in matrix were taken as the horizontal coordinate, and the ratio of the two to the IS peak area was taken as the vertical coordinate. The weighted least square method was used to perform regression operations, and the calibration curves of lacosamide and ODL were obtained respectively. LLOQ is the minimum concentration of the calibration curve and should be accurate and precise in the range of ±20%.To achieve inter-day and intra-day precision and accuracy, QC samples were analyzed in five batches over three consecutive days. Separate calibration is performed on each verification day. Relative standard deviation (RSD%) is used to indicate accuracy, and relative error (RE%) is used to indicate accuracy. Accuracy of ±15% or less, precision of 15% or less is considered acceptable.Lacosamide and ODL standard solutions were added to the treated various blank matrices to prepare three QC concentrations. The peak areas obtained were compared with those obtained from three QC levels of pure standard solutions, that is, matrix effect. The recovery was obtained by comparing the peak area before extraction of lacosamide and ODL with the peak area after extraction at the three QC levels.Three concentrations of QC matrix-match samples were placed in various conditions to test the stability of lacosamide and ODL for short-term, long-term, three freeze-thaw cycles, and at autosampler. Sample storage at room temperature for at least 3 h was used to test the short-term stability. After 21 days of storage at −80°C, samples were tested to establish their long-term stability. By assessing the stability of three freeze-thaw cycles from freezing (−80°C) to thawing (room temperature) three times, the stability of the samples was assessed. Additionally, the stability of the produced samples was assessed after 4 h of storage in an autosampler at 10°C.m (Michaelis-Menten constant) sequence concentrations. Incubation system of 200 μL was made up of 100 mM of PBS buffer, 0.2 mg/mL of RLM, 1 mM of NADPH, and 20–2000 μM of lacosamide. In addition to NADPH, the solution was mixed and pre-incubated at 37°C for 5 min prior to the addition of 1 mM NADPH, followed by incubation for 50 min and placed at - 80°C to discontinue the reaction. After complete completion of the enzyme reaction, the mixture was added 10 μL of 200 ng/mL IS solution and 300 μL of acetonitrile (protein precipitant). The mixture was then swirled for 2 min and centrifuged at 13,000 rpm for an additional 10 min, and the supernatant of 100 μL was placed in an injector for the quantification by UPLC-MS/MS.The range of 20–2000 μM of lacosamide had been dissolved in DMSO to comply with the Km of lacosamide was 1,016 μM. In order to investigate the potential DDIs of lacosamide, the 200 μL system remained unchanged, and using Km as the concentration of lacosamide in the RLM system, 7 cardiovascular drugs  were tested for inhibition on the metabolism of lacosamide. The following reaction procedure was identical to the above-mentioned enzyme reaction. The experiment had to be redone with an inhibition rate of 80% or higher.In the RLM incubation system, the K50) of inhibitors  against lacosamide was determined in RLM. The cardiovascular drugs was dissolved in DMSO and diluted with DMSO at a concentration gradient of the IC50 was 0, 0.01, 0.1, 1, 10, 25, 50 and 100 μM, and the concentration of lacosamide was 1,016 μM in RLM. The subsequent processing steps were the same as above to obtain the IC50 values of inhibitors in RLM. To identify the mechanisms underlying the inhibitory effect of nisoldipine on lacosamide, 0, 1.7, 3.4 and 6.8 μM of nisoldipine , and 254, 508, 1,016, and 2032 μM of lacosamide  in the RLM system, were selected. The subsequent reaction and treatment process were the same as the described above.The half maximal inhibitory concentration (ICn = 6): group A (control group) and group B, C . The animals fasted for 12 h before the experiment, with no restricted water intake. Lacosamide, felodipine, and nisoldipine were prepared with suspension of 0.5% sodium carboxymethyl cellulose (CMC-Na) solution and administered orally. Groups B and C were given felodipine (1 mg/kg), and nisoldipine (1 mg/kg) by gavage, respectively, and the same amount of CMC-Na solution was administered to group A. After 30 min, all rats were received 10 mg/kg of lacosamide by gavage. Tail venous blood was collected at 0.333, 0.667, 1, 1.5, 2, 3, 4, 6, 8, 12, 24 and 48 h after dosing. The blood sample (0.3 mL) was centrifuged at 8,000 rpm for 5 min 100 μL of plasma was obtained and frozen at - 80°C for further processing. The subsequent treatment was carried out according to the chapter of Pre-treatment of samples.18 Sprague-Dawley (SD) male rats (200 ± 10 g) were bought from the Animal Experimental Center of the First Affiliated Hospital of Wenzhou Medical University and were randomly assigned to three groups  software  with non-compartment model analyses was used to obtain the pharmacokinetic parameters of lacosamide and ODL in rats. The comparison of the pharmacokinetic parameters among three groups in rats was performed with SPSS , with one-way ANOVA, and the p-value <0.05 was regarded as statistically significant.The GraphPad Prism 9.0 software was used to generate the Kr2 = 0.996); ODL in plasma, y= ; ODL in RLM, y= . The calibration standard curves illustrated excellent linearity at a concentration range of 2–10000 ng/mL for lacosamide and that of 1–1,000 ng/mL for ODL in plasma, also 80–40000 ng/mL for ODL in RLM.The method for the quantification of lacosamide and ODL had been successfully established, as illustrated in Four concentration levels of each analyte have been employed to measure the precision and accuracy. Lacosamide and ODL have intra-day and inter-day precision of less than 15% and accuracy of ±15% at three QC levels. As for LLOQ, the precision was below 20% and the accuracy was within ±20%. All the results in various matrices are presented in Three QC levels were used to study the recovery and matrix effects. As shown in The stability of lacosamide and ODL in various matrices was determined by the stability test, and were expressed in m value for lacosamide was determined by non-linear regression of the rate of reaction to the concentration of the substrate  with inhibition rates greater than 80% were screened out . The IC50 was below 10 μM, suggesting a modest inhibition of the metabolism of lacosamide in vitro. The inhibitory mechanisms of nisoldipine on lacosamide in RLM were further investigated, and it inhibited the metabolism of lacosamide with a Ki of 2.886 and αKi of 2.941, by a mixed inhibition mechanism, involving un-competitive as well as non-competitive inhibition, in accordance with the Lineweaver-Burk plots (The ICrk plots .(0-t), AUC(0-∞) and Cmax of lacosamide were significantly increased when nisoldipine was administered in combination with lacosamide in rats, while CLz/F, Tmax, and, Vz/F were significantly decreased. And, there was no significant change in MRT(0-t), MRT(0-∞) and t1/2z. These results suggested that nisoldipine could increase the plasma exposure of lacosamide in SD rats, demonstrating that nisoldipine might be a potential interaction drug with lacosamide. With respect to ODL, the AUC(0-t), AUC(0-∞) and Cmax increased significantly, the MRT(0-t), MRT(0-∞), CLz/F and Vz/F decreased significantly, and t1/2z and Tmax had no significant difference when nisoldipine was administered. As for felodipine, it did not change the exposure of lacosamide and ODL in rats.The average concentration-time curves of lacosamide and ODL in rats are shown in People with epilepsy often have complications or underlying conditions that may be combined with cardiovascular medications . Dependim/z 251.1→108.2,m/z 237.1→108.2 and m/z 256.0→145.0, respectively. Samples need to be processed before UPLC-MS/MS detection, and we used acetonitrile one-step precipitation extraction to reduce interference from endogenous substances, especially proteins, and to obtain permitted recovery and matrix effects.To better analyze lacosamide, ODL, and IS and produce more precise analytical results, the MS settings were improved in this experiment. Lacosamide, ODL, and IS have good peak and separation effects following the optimized gradient elution process with 0.1% formic acid in water and acetonitrile as mobile phase, and the retention time is 1.18 min, 1.12 min and 1.14 min, respectively. The quantitative fragment ions of lacosamide, ODL and IS were in vivo study, 5 drugs with higher inhibitory rates were selected for their IC50 values, all of which were less than 10 μM, and 2 drugs  were selected for in vivo pharmacokinetic experiments in rats. All two cardiovascular drugs are calcium channel blockers that are widely used in the treatment of cardiovascular disease and are clinically approved for the treatment of hypertension . These results suggested that nisoldipine may exhibit mixed inhibition through multiple pathways.As far as we know, nisoldipine is a substrate of CYP3A4, but its inhibitory effect on CYP450 has not been reported. In this study, we firstly studied the inhibitory mechanism of nisoldipine on lacosamide in RLM through the production of ODL 1/2z, CLz/F and Vz/F of lacosamide alone in rats were similar to those in our study, with slight differences in Cmax and Tmax, such differences may have resulted from individual variances between rats. The values of AUC(0-t) and MRT(0-t) are roughly half of those in this study, which are 9270.3 ± 3231.7 ng/mL*h and 3.6 ± 0.5 h respectively (max (0.459 μg/mL) in healthy subjects was consistent with our study of Cmax (558.0 ± 220.5 ng/mL)  .max were increased significantly, and CLz/F were decreased by about 50%, so the metabolism of lacosamide were slowed down and/or drug absorption were increased. Nisoldipine had no significant effect on the final t1/2z of lacosamide, indicating that nisoldipine had a moderate effect on lacosamide clearance, and the effect on the final t1/2z was not as significant as that on AUC. The AUC, Cmax, and CLz/F values for ODL were consistent with the prototype in the presence of nisoldipine, according to the pharmacokinetic data of the metabolite. This result could be explained by lacosamide’s higher bioavailability in the presence of nisoldipine, which suggests that greater amounts of lacosamide has the potential to be transformed into ODL. However, in vivo studies in rats cannot properly predict the situation in people since the role of the CYP450 enzymes for substrate metabolism in humans and rats differ, and additional research is required to confirm this in clinical investigations.The results of pharmacokinetic experiments in rats showed that in the present of nisoldipine, AUC and Cin vivo and in vitro. Due to the wide range of clinical applications of lacosamide and nisoldipine, we could provide basis for the appropriate and rational combination of lacosamide and nisoldipine, offer evidence for the joint development of antiepileptic drugs and cardiovascular drugs, and furnish support for personalized clinical medication.All in all, this study assessed the impact of DDIs on the metabolism of lacosamide. Through sample pre-treatment and complete methodology verification, the method was characterized by simple operation, high recovery, high sensitivity and specificity. When co-administered with lacosamide in rats, nisoldipine could significantly alter lacosamide and ODL pharmacokinetic parameters. In addition, we found that the inhibition of nisoldipine on lacosamide was consistent


Callitriche cophocarpa Sendtn. belongs to a genus with a potential for accumulation of elevated metal levels. Thus, it may provide consolidated evidence of contamination. Therefore, the aim of this investigation was to determine Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn in this species collected together with water and bottom sediments from rivers with various levels of pollution. Of these rivers, one less polluted and one more polluted was selected for the collection of C. cophocarpa for an experiment to compare its Cu and Zn concentration potential. Both metals were supplemented at concentrations 0.01, 0.02, 0.03, 0.05, 0.08 and 0.14 mg L−1 of Cu as CuSO4 × 5H2O and 0.4, 0.6, 0,9, 1,35, 2.03 and 3.04 mg L−1 of Zn as ZnSO4 × 7H2O, and in the binary design containing (mg·L−1) 0.01Cu + 0.4Zn, 0.02Cu + 0.6Zn, 0.03Cu + 0.9Zn, 0.05Cu + 1.4Zn, 0.08Cu + 2.03 Zn and 0.14Cu + 3.04Zn. The upper concentrations of Cr, Cu, Mn and Zn in C. cophocarpa shoots from both types of rivers as well as of Ni and Pb in shoots from more polluted rivers were higher than the values typical for toxicity thresholds with no visible harmful effects, which may indicate accumulation abilities of C. cophocarpa for these metals. Both roots and shoots of C. cophocarpa may be included in the group of macroconcentrators for bottom sediments with respect to Cd, Co, Cr, Cu, Fe, Mn, Ni and Zn and deconcentrators of Pb. Greater accumulation of most metals in roots than in shoots indicates their restricted mobility and translocation by C. cophocarpa to shoots. C. cophocarpa from the less polluted river and exposed to all experimental solutions contained significantly higher levels of Cu and Zn than that from the more polluted river exposed to identical experimental solutions. The plants collected from the more polluted river influenced by surplus of metals and living under chemical stress could probably limit further accumulation by developing a resistance mechanism. Cu and Zn contents in C. cophocarpa were higher when treated with separate metals than for binary treatment both in the more and less polluted river. Such research presenting the impact of a combination of metals could be important for understanding and explaining the interactions of these elements which may influence their bioavailability in nature as well as importance in the evaluation of the risk of environmental toxicity.The anthropogenic impact of metals on aquatic environments is a risk for biota, and thus their levels must be controlled. The online version contains supplementary material available at 10.1007/s11356-023-28372-5. Callitriche cophocarpa Sendtn. belongs to a genus with a potential for accumulation of elevated metal levels. For instance, C. stagnalis was reported as a good accumulator of U reflecting the natural load of this element in mineralised areas  after their single or combined application. The tested hypotheses were: binary Cu and Zn concentrations influencing C. cophocarpa may cause mitigating effects on the content of these metals in the species; C. cophocarpa collected from the more polluted river accumulates significantly more Cu and Zn than the same species from the less polluted river because it has developed adaptation to elevated levels of both elements.Contamination of aquatic habitats is a crucial problem of ecological interest because of its harmful influence on living organisms, their biodiversity and water cleanliness . Bottom sediments were homogenised using an agate mortar with a pestle after removing any coarse material with a 2-mm sieve. Plant-available Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn in bottom sediments were assayed by extraction with 1 M ammonium acetate-EDTA (pH 4.65) for 30 min (10 g dry soil in 100 mL) . After dilution to 50 mL, the digests together with water samples were analysed for Fe, Mn and Zn using FAAS (Avanta PM from GBC) and Cd, Co, Cr, Cu, Ni and Pb using GFAAS (PinAAcle 900Z Perkin-Elmer Graphite Furnace Atomic Absorption Spectrometry). All the elements were determined against standards , and blanks containing the same matrix as the samples and were subjected to the same procedure. All analyses were done in triplicate, and all results for plants were calculated on a dry weight basis. The accuracy of the methods applied for the determination of the elements in the samples was checked by the analysis of Chestnut Soil, Bainaimao and Bayan Obo, Neil Mongol in China GBW07402 (GSS-2) and Poaceae (mixture) IPE 952WEPAL Certified Reference Materials  and potentially polluted (no. 3) river sites  0.01Cu + 0.4Zn, 0.02Cu + 0.6Zn, 0.03Cu + 0.9Zn, 0.05Cu + 1.4Zn, 0.08Cu + 2.03 Zn and 0.14Cu + 3.04Zn  on Box-Cox transformed data was applied to calculate the differences among the metal concentrations in water, sediments and C. cophocarpa between the investigated rivers . The post hoc Tukey test was used to compare Cu or Zn contents in experimental C. cophocarpa when Cu, Zn and Cu + Zn were added  were compared with the t-test n1/. The general toxicity condition of the sample: PLI ≤ 1 shows baseline levels of pollutants while its growth to 1 < PLI shows progressive deterioration of aquatic ecosystem quality  or a plant-available metal in bottom sediments (BFs)  by Kabata-Pendias  of less polluted rivers were significantly lower than in more polluted rivers  were estimated ESM –10. In pC. cophocarpa may be included in the group of macroconcentrators (BFs > 2) for Cd, Co, Cr, Cu, Fe, Mn, Ni and Zn and deconcentrators for Pb (BFs < 1). Probably, this species restricts accumulation of Pb as a metal not essential for plants, toxic even in very low concentrations . On day 7, C. ons Fig. . Such meons Fig. . Metal-rons Fig. . Chelations Fig. . Some plons Fig. . C. coph−1 Cu and 16,210 mg kg−1 Zn (from the less polluted river) with no visible symptoms of harmful effects Supplementary file2 (PDF 22 KB)Supplementary file3 (PDF 230 KB)Below is the link to the electronic supplementary material.