Full Text: 1 Introduction The pulp and paper mill is one of the major pollution-causing industry in the world due to the presence of different types of organic, inorganic, and metallic pollutants in the effluent (Singh & Chandra, 2019). It generated 150-200 m³ wastewater (effluent) per ton of paper production (Pokhrel & Viraraghavan, 2004). The discharged wastewater contains 40-45% raw materials which are loaded with lignocellulose, chlorolignins, fatty acids, chlorinated resins, chlorinated phenols, chlorophenols, pesticides, and biocides i.e., collectively known as organic pollutants (Lindholm-Lehto et al., 2015). These organic pollutants may be mutagenic, carcinogenic, endocrine-disrupting, and clastogenic due to high biological oxygen demand (BOD), chemical oxygen demand (COD), total dissolved solids (TDS), and total suspended solids (TSS) in wastewater  (Karrasch et al., 2005; Yadav & Chandra, 2018). Besides these, some trace elements are also present in the effluent limit which can cause toxicity in crops. The organic component of effluent provides natural media for the growth of pathogenic microorganisms which is also harmful to humans (Chandra et al., 2006; USEPA, 2012). However, toxic effects of the effluent in the natural ecosystem are also noticed such as color problems, thermal impacts, scum formation, slime growth, and loss of biodiversity (Zhang et al., 2013; Jaramillo & Restrepo, 2017). It may be a dangerous sign to the survival of animals and crops. The refractory chemical compounds are also released during the chemical bleaching and paper making process i.e. chlorolignins, chlorophenols, and biocides are persistent in nature and bio-accumulate in the fatty tissues of the tubifex worms which are characterized as lipophilic compounds, that are capable to easily penetrate the cell membrane (Lindholm-Lehto et al., 2015). The derivatives of effluent chemical compounds are accumulated in the plant and tubifex-tubifex worm and also reached into the trophic level via the food chain into the natural ecosystem (Raj et al., 2007). However, the chlorophenols derivatives cause adverse effects on the food chain in the environment and the adverse effect was noticed on the primary, secondary and tertiary consumers (Colette & Kevin, 2017). The Organization for Economic Co-operation and Development (OECD) guideline the wheat and mustard crops are valuable worldwide for cultivation in the agricultural field. Hence these plants are used as model test crops in research. However, the knowledge of the refractory organic pollutants of paper mill effluent is not much known.  Hence, this article provides clear and advance knowledge about the toxic properties of chemicals released with effluent from the paper industry. However, there are many treatment methods are available (i.e. physical, chemical, and biological) for reduction and remediation of color and toxicity of the effluent but, in these processes high molecular weight chemical compounds, toxicants, dissolved solids, suspended solids, and low molecular weight compounds are not removed properly and method is highly cost-effective (Hossain & Ismail, 2015). Besides, low molecular weight compound contains chlorinated ring structure in pentachlorophenol (PCP) it is anticipated to be recalcitrant to aerobic biodegradation in running wastewater. Whereas, aromatic compounds with greater chlorine levels are usually highly resistant towards biodegradation and biotransformation. According to the US Environmental Protection Agency (USEPA, 2012), PCP is a priority pollutant and is found higher than the permissible limit (0.30 μg/l^-1) even after tertiary treatment. Pulp paper mill effluent contains heavy metals that cause a toxic effect in the crop plants as well as heavy metals accumulate in the roots, stems, and leaves and also stored in the seeds of the plants and affect the food chain (Reza et al., 2015). The present study has been focused on the detection of organic pollutants released from pulp paper mill and their toxicity has been checked on T. aestivum and B. campestris (terrestrial model) and Tubifex-tubifex (an aquatic model organism). It showed pulp paper mill effluent cannot be useful for irrigation due to their toxic properties in the environment.   2 Materials and methods 2.1 Sample collection and analysis The effluent and sludge samples were collected from the Yash paper mill Faizabad (U.P.), India and kept at 4⁰C icebox container for further analysis. 2.2 Physico-chemical analysis of the pulp paper mill effluent The Standard for the examination of water and wastewater standard methods (APHA, 2005) was followed for the analysis of physicochemical parameters of effluent and sludge samples. Physico-chemical parameters i.e. biological oxygen demand (BOD), total dissolved solids (TDS), chemical oxygen demand (COD), phosphate, total phenol, total nitrogen, and sulfate were analyzed (APHA, 2005). The color was measured by using a Canadian pulp and paper Association conventional technique at 465 nm by UV–visible spectrophotometer 2300 (Evolution 201) (CPPA, 1994), pH of the sample was evaluated using a pH meter (Water and Soil Analysis Kit ESICO 1160) and dissolved oxygen was measured using a Clark polarographic dissolved oxygen sensor (835 A, Germany) 0.1 mg/l. Lignin content was measured as per the Pearl & Benson (1940). Heavy metals and various ions such as chloride, sodium, potassium, and nitrogen were estimated by atomic absorption spectrophotometer (AAS) and by the selective ion electrodes (Orion Model 960). Sugar content was estimated by the phenol sulfuric acid method (Chow & Landhausser, 2004). 2.3 Extraction and identification of various refractory organic compounds The organic refractory compounds present in effluent and sludge were extracted by dichloromethane (DCM) at pH 8.0. An aliquot of the concentrate was dissolved in 3.0 mL DCM, filtered through 0.22-μm syringe filters, and used for further Gas-chromatography and mass spectrometry (GC-MS) analysis. 2.4 Gas-chromatography and mass spectrometry analysis of refractory organic pollutants Before the characterization of the refractory organic pollutants, various organic solvents i.e., acetone, dichloromethane, n-hexane, methanol, and isopropyl alcohol, were tested to compare the ability to extract residual organic pollutants, and dichloromethane was found to be optimal. The GC-MS analysis of the dichloromethane extracts was performed following the method described by Chandra et al. (2009) for the characterization of the residual organic pollutants. The residual organic pollutants were identified by comparing their mass spectra with those provided in the National Institute of Standards and Technology (NIST) library, which was supplied with the instrument. 2.5 Toxicity assessment by pot experiment test To analyze organic pollutant toxicity in T. aestivum and B.campestris using pulp paper mill wastewater and sludge. A pot experiment was conducted at the research field of Bioremediation and Metagenomics under the greenhouse condition in the Department of Environmental Microbiology,  Babasaheb Bhimrao Ambedkar University Lucknow, India, 226025. Thirty-two pots were filled with three kg of soil mixed with a sludge sample.  The wheat and mustard variety (UP-2338 and B-17) were sown in eight sets each set contains four pots, two control sets (filled with agricultural soil one set for UP-2338 and second forB-17). While the remaining six sets were treated with various amount of pulp paper mill sludge amended with agricultural soil. Twenty seeds of selected wheat (UP-2338) and mustard variety (B-17) were sown in each pot. However, the remaining six sets were filled with different concentration of sludge with an agricultural soil viz., (i) 25% sludge and 75% agricultural soil; (ii) 50% sludge and 50% agricultural soil; (iii) 75% sludge and 25% agricultural soil, and (iv) 100% sludge. Three replicates of the selected crop were made for each treatment. Plants were irrigated through pulp paper mill wastewater as per requirement. Different morphological parameters of different varieties of wheat and mustard were also observed. The maturation period for both crops was 5 months, so plants were harvested in April 2019. 2.6 Tubifex-tubifex toxicity Tubifex worms were collected from Gomati River, Lucknow, Uttar Pradesh India from natural sources and acclimatized in laboratory conditions for 7 days before the experiment. Ten tubifex worms were exposed for 96h of each concentration (25%, 50%, 75%, and 100%) through direct effluent sample and 0% (tap water) used as control. When they were fully immobilized and no reaction pressed with a blunt glass rod, test worms were regarded to be dead. Death was further verified by returning worms to tap water with new control. Every 24 hours, test water was renewed and the experiment was conducted in triplicate. 2.7 Statistical analysis For the statistical analysis, of variance (Twoway ANOVA) by student T-test statistics and means were compared by Duncan’s multiple range test (DMRT) (Duncan, 1955). 3 Results and discussion 3.1 Physico-chemical parameters Physicochemical parameters of paper mill effluent showed dark brown color, foul odor, and turbid. It might be the presence of polymeric lignin degradation products formed during the black liquor stage of pulping(Chandra & Abhishek, 2010; Chandra & Yadav, 2017). Color in the effluent is usually associated with aromatic compounds produced from the decay of organic pollutants which were released from the paper industry. However, dark colours can cause the problems of both water opacity and the blanketing of the river. The dark colour and blanketing can reduce photosynthetic activity in aquatic plants (Singh & Thakur, 2004). This leads to a chain of adverse effects on the aquatic ecosystem as the growth of primary consumers as well as secondary and tertiary consumers are adversely affected (Ruggicro et al., 1989). The color of effluent also depends on pH and it was recorded slightly alkaline (7.89±0.53). Slightly alkaline pH was the result of the bleaching process during paper manufacturing and it was not beyond the permissible limit (Livernoche et al., 1983). The low pH values could be the result of organic acids caused by the fungal metabolism and the high pH level is the result of paper bleaching (Holt et al., 2008). The concentration of BOD (788±3.16 mg/l⁻¹) and COD (416±2.13 mg/l⁻¹) was observed high in the effluent. The high amount of BOD and COD value in effluent indicates the presence of organic and inorganic-organic pollutants in the effluent (Yadav & Chandra, 2018). It might be due to the presence of cellulose, hemicelluloses, lignin, xylose, chlorinated phenolic compounds, and other related compounds used in the bleaching and wood digestion process. During wood digestion sodium hydroxide and sodium sulfide were used for fibers separation in the dissolving tank. After that, fibers removed and the remaining water was treated as black liquor. Due to the caustic nature of black liquor, it is expected that there would be toxic effects (e.g. edema) to the respiratory system if mists or vapors are inhaled and toxic effects for skin and eye exposures, especially at elevated temperatures (Singh & Chandra, 2019). The toxicity ranking above is associated with representative components of the black liquor (sodium sulfide and sodium hydroxide, etc.) which are presented as a determination of acute toxicity of complex mixture by analogy. The chloride (1942±8.45 mg/l⁻¹) and sulfate (136±2.21 mg/l⁻¹) ions were found to be at optimum level and were generated from bleaching and sodium sulfite processes (Singh & Chandra, 2019). One particular study, conducted by Wurtz & Bridges (1961), including two of the four species suspected of being most sensitive to chloride (a Iowa DN planorbid snail, Gyraulus circumstriatus, and the fingernail clam, Sphaeriu mtenue). Further, Khangarot (1991) suggested acute chloride toxicity data for the tubificid worm (Tubifex-tubifex), which indicated that this species might also be highly sensitive to chloride. Phosphate (70.86±1.16) was found below the permissible limit in the effluent sample and higher in the sludge (1223.62±46.47) and garden soil (886.78±16.53). The same results were also reported by Singhal & Thakur, (2009). Phosphate has the potential to cause increased algal growth leading to eutrophication in the aquatic environment. However, there is no adequate information available on risk assessment or acute and chronic toxicity. The elevated level of chemical parameters in the effluent due to lignocellulosic raw materials used in the manufacturing process wood digestion, pulping, bleaching, paper making, and chemical recovery (Chandra & Singh, 2012).The heavy metals were recorded as Cd (0.618 ± 0.12 mg/l⁻¹), Cu (2.156 ± 0.22 mg/l⁻¹), Fe (6.384 ± 0.89 mg/⁻¹), Ni (2.148 ± 0.21 mg/⁻¹), Pb (0.265 ± 0.11 mg/⁻¹), and Zn (3.245 ± 1.51 mg/^-1) was recorded in the paper mill effluent. All metals were found at an optimum level as per the permissible limit except iron. Due to heavy metals, serious health hazards are caused due to the transfer of these contaminants into the food chain (Singh & Chandra, 2019). Due to changing environmental conditions and extreme use of agrochemical heavy metals in the paper industry are being accumulated in soils that are transferred to the water system by leaching. This poses a serious threat to human life and the environment (Nicholson et al., 2003; Wong et al., 2003). However, the ingestion of large quantities of iron results in hemochromatosis. It is a condition in which normal regulatory mechanisms do not operate effectively which leads to tissue damage as a result of the accumulation of iron. The details of the analyzed physicochemical parameters were shown in table 1. 3.2 Characterization of refractory organic pollutants from pulp paper mill effluent The extracted effluent and sludge samples were characterized through showed various peaks at different retention times (RT) (figure 1.a, b). In the effluent sample, major peaks recorded at RT 27.33 and 43.52,were characterized as docosane; tetradecane; hexadecaneand 1,8-diphenyl-3,4,10,11-tetrahydro[1,4]dioxino[2,3-g:5,6-g']diisoquinoline,  3-á-acetyl-7-azidocholesterol, respectively (table 2). However, minor peaks were observed at different RT7.80, 10.37, 12.89, 17.47, 21.54, 25.18, 31.30,  37.06, and 48.15 and interpreted as the hexadecane, 2-methyl-, ; methyl 1-iodomethyl-2-oxocyclotetradecanoate; eicosane, heptadecane, octadecane,  pentadecane;  tricosane, eicosane, dodecane; dodecane, hexadecane, octadecane; docosane,  tetradecane,  hexadecane; pentachlorobromobenzene, rans, trans-alloocimene; 9-(1,1-(2)H(2)-ethyl) anthracene, bis(3,4-dimethoxycinnamoyl)-L-tartaric acid, 4-Cyano-2-methyl-N-phenylacetanilide and 5,11,17,23, tetrakis (1,1dimethylethyl) 28methoxypentacyclo [19.3.1.1(3,7).1(9,13).1(15,19)] octacosa1(25),3,5,7(28),9,11,13(27),15,17,19(26),21,23-dodecene-25,26,27-triol, respectively in the effluent (Table 2). These organic compounds were also generated by degradation, migration, and transformation processes during water movement after released from the industry (Yadav & Chandra, 2018). However, larger molecules of organic matter can be formed from the polymerization of different parts of the already broken down matter. The composition of natural organic matter depends on its origin, transformation mode, age, and existing environment, thus its bio-physicochemical functions vary with different environments (Nicola et al., 2006). The hexadecane was detected in the GC-MS analysis from the effluent sample which is used as biocides in the paper industry and kraft recovery process. This compound can cause different problems to humans such as lung irritants, endocrine-disrupting chemicals, headache, dizziness, tiredness, nausea, vomiting, depression, and gastrointestinal tract irritation (Singh & Chandra, 2019). Decane, undecane, and dodecane were recorded for cytotoxicity at high concentrations. However, tetradecane, pentadecane, and hexadecane were reported for hematotoxicity. The mixed solvent of chemical compounds was toxic to human epidermal keratinocytes (Yang et al.,2006). The organic pollutants octadecane, pentachlorobromobenzene, tricosane, and methyl 1-iodomethyl-2-oxocyclotetradecanoate, may also be produced from the pulp bleaching and biocide used as a preservative which has toxic properties, it may act as endocrine-disrupting chemicals and also very toxic to aquatic life (Yadav & Chandra, 2018). The other pollutants may also report for causes toxicity to the environment which is listed in table 2. However, the refractory organic pollutants that were detected in the sludge sample showed a measure peak at RT 6.17, 3-mercaptobenzoic acid, tertbutyldimethylsilyl ester compounds were detected and released from the wood extract during the digestion process. It may cause eye, skin irritation, and respiratory problems. The compound octadecane; hexacosane; tetradecane was detected at RT 7.79 which was generated from the bleaching process and have Endocrine-Disrupting chemical properties (EDCs). The octadecane properties of EDCs were also reported (USEPA, 2012; Yadav &   Chandra, 2018). However, pentasiloxane, dodecamethyl- was detected at RT 10.37 and released from the bleaching and paper making process that causes toxicity to the aquatic ecosystem and accumulate in fish and other organisms through the food chain. The chemical compounds tricosane; hexadecane, 2-methyl-; octadecane, 1-iodo- was detected at RT 21.52 which was released from the paper industry and these were used as biocides in the paper preservation process. It may cause skin problems (irritant, sensitizer, lung irritant) and lung cancer also. The oleic acid, trimethylsilyl ester; trans-9-Octadecenoic acid, compound were also detected in GC-MS at RT 29.84 and they are generated from the plant essential oil. The acute exposure was noticed due to plant essential oils such as the central nervous system (CNS) depression, and gastrointestinal tract irritation. However, tetratetracontane; nonacosane compound was found at RT 38.61 that causes headache, nausea, vomiting, diarrhea and abdominal pain which were generated from the bleaching process of the paper mill (Yadav & Chandra, 2018). The compound 1,8-Diphenyl-3,4,10,11-tetrahydro[1,4]dioxino[2,3-g:5,6-g']diisoquinoline; 3-á-Acetyl-7-azidocholesterol was found at RT 43.51 which was used as biocides in paper making process causes asthma to a human being. The organic compound meso-2-Nitro-5,10,15,20-tetra (4-pyridyl) porphyrin; 1,4- Bis [3,5-bis-trimethylsilylethynylphenyl) ethynyl] benzene was detected at RT 48.12 from the paper industry which was released after the bleaching process in the effluent that causes acute toxicity; acute exposure to hexadecane causes irritation, CNS depression, and gastrointestinal tract irritation (Kim et al., 2016). However, some other compounds were also detected which were shown in table 3. Above mentioned toxic organic compounds were released directly into the environment as effluent that has different toxic properties which are listed in USEPA, 2012. These compounds leached out into the groundwater which causes health hazards in human beings. 3.3 Pot experiment test The toxicity of effluent and sludge was evaluated based on percentage germination and toxicity percentage as per the method followed by Oljira et al. (2018). The growth parameters of plants i.e. germination percentage; root length; shoot length; leaf number and total dry weight produced was measured immediately with the help of the centimeter scale after harvesting (Subramani et al., 1997; Ramana et al., 2002). The plant was dried at 72⁰C for 72 h for dry weight estimation (Wong et al., 1996). According to Arnon’s method (Arnon, 1949), chlorophyll content was estimated of both treated and untreated (control) fresh leave samples by spectrophotometer. After secondary treatment of effluent, it was reported to cause significant effects on seed germination parameters due to its potentially toxic nature as revealed by physicochemical characterization. The result of germination as well as seedling growth of T.aestivum and B.campestris seeds in different concentrations (25, 50, 75, and 100%) of sludge sample with agricultural soil and irrigated by effluent as presented in table 4. The seedling growth compared with control, root lengths and shoots of seedling were highest at 25% (w/w) sludge and afterword gradually decreased with increasing sludge concentrations. The phytotoxicity was maximum at higher sludge concentration and minimum at lower sludge concentration. This might be associated with the presence of highly toxic complex organic pollutants, toxic metals in pulp paper mill effluent, and sludge. The toxicity of sludge might be responsible for inhibiting the growth parameters. It has been reported that the various highly toxic complex organic pollutants along with metals act as an inhibitor for several phytohormones, such as alpha-amylase, gibberellins, auxins, and cytokinins, which act a crucial role in seed germination (Chandra et al., 2011). Additionally, significant changes were observed in several physiological parameters and revealed more accurate information about the harmful effects of pulp paper mill effluent and sludge in the pot experiment. The different concentrations of pulp paper mill sludge significantly (P>0.05) contributed towards the percent (%) phytotoxicity of the pot experiment (figure 2). 3.3.1 Estimation of morphological parameters In test plants, T.aestivum and B.campestris were tested at different concentrations of the sludge sample. The plant growth parameters (i.e. root length and shoot length) showed a significant decrease as compared to the control plant beyond above 50 % (w/w) concentration shown in table 5. The 25% (w/w) concentration of paper mill sludge sample was tolerated by the T. aestivum and B. campestris plants and maximum increase in root length, shoot length and total dry weight were observed. Moreover, the T. aestivum and B. campestris plants treated with 75% (w/w) pulp paper mill sludge   have shown vigorous growth initially but later reduced growth, delayed flowering and fruiting was observed as compared to the control. The different concentrations of pulp paper mill sludge significantly (P>0.05) contributed towards % phytotoxicity of root and shoot. The inhibition of seed germination and plant growth at higher concentrations of sludge-amended soils may be due to the high salt concentrations, toxic pollutants in pulp and paper mill sludge creating high osmotic pressure and toxicity. The high osmotic pressure and pollutant toxicity caused inhibition of seed germination and their growth parameters. Similar observations have been reported earlier by Chandra et al. (2008). 3.3.2 Effect on photosynthetic pigments The photosynthetic surface area and leaf chlorophyll content were the key factors for determining dry matter production of T. aestivum and B. campestris. There was an increase in chlorophyll-a, complete band chlorophyll content in leaves T. aestivum and B. campestris plants at 30 and 60 days of development in different concentration of pulp paper mill effluent and sludge. However, the pigment concentration decreased significantly above 50% concentration of sludge after 60 to 90 days of growth period as compared to                the control as shown in table 6. The highest photosynthetic pigment  (chlorophyll-a, band total chlorophyll) formation was noticed up to 25% (T. aestivum and B. campestris plants) at 60 days of the growth period and that was compared with control. Due to continuous increases in sludge concentrations in pot, significantly photosynthetic pigments were decreased in the plant. However, these metals, which were acting as nutrients crossed the threshold limit at high sludge concentrations and work as toxic agents through direct inhibition of photosynthesis (Chandra et al., 2008). 3.4 Toxicity of paper mill effluent on Tubifex-tubifex Laboratory toxicity testing was also an essential tool for assessing the potential effect of chemicals on ecological systems through environmental evaluation (Shuhaimi-Othman et al., 2012a). Several freshwater pollution evaluations and toxicity testing were recorded with ligochaetes, particularly from the tubifex and Lumbriculidae families, such as Tubifex-tubifex and Lumbriculus variegates (Khangarot, 1991; Mosleh et al., 2007; Sardo & Soares, 2011). The high sediment contamination of different concentrations of paper mill effluent triggered the autotomy process and showed a clear reduction in growth rate and other activity of tubifex worm. This study showed the toxicity of different concentrations (25%, 50%, 75%, and 100%) of paper mill effluent compared with control (tap water) shown in figure 3 and table 7. During the test period, tubifex worms have remained active in control. At the bottom of the test container, they were clustered and showed typical tubifex movement. At the starting of the experiment, test animals remained separated in the higher concentrations of pulp paper mill effluent and showed rapid twisting movement. The later intoxication phase was the worm's reduced tactile movements. The body was segmented and degenerated, and death occurred without any other noticeable signs as it may be due to the presence of chlorinated toxic compounds in the effluent. The causes of toxicity of high concentration of heavy metals were accumulated in body parts of the worms (Shuhaimi-Othman et al., 2012b). After 24 hours of exposure, morphological changes, hemoglobin content disappeared at the lethal concentrations of pulp paper mill effluent, cell bursting, and the back of the body became white, with body disintegration were observed. In general, disintegration begins from the back of the body and progresses to the front. However, these similar results in tubifex worms were also reported by Khangarot, (1991). Due to the rapid increase in the concentration of paper mill effluent the worms getting dead. Conclusions In the present study, toxic organic pollutants were detected from the pulp paper industry. The discharged effluent contains different chemicals that have endocrine-disrupting properties. This is the first report on the detection of persistent organic pollutants and their toxicity in agricultural crop and aquatic ecosystems. However, due to the presence of organic chemical compounds in the affluent, there is an increase in the BOD, COD, and TDS level in the aquatic resources and the aquatic organisms (Tubifex-tubifex) get affected in terms of their reproduction and other metabolic activities were stopped and they may be lost or died. Besides, these chemicals may also increase the toxicity level in the receiving water sources. The crop plants (T. aestivum and B. campestris) are also affected due to the excessive use of sludge and effluent in the crop field as irrigated water in undeveloped areas where there is no proper irrigation system available. The affected crop has been destroyed by its root length, shoot length, and seed quality. The chemical pollutants of the effluent may also reach the body of higher animals through the food chain. The effect of effluent was noticed and found roots of the plants were reduced and tubifex worms cell wall became whitened and burst in above 25% concentration of effluent and sludge. Hence, this research article is very useful for the researchers for the understanding of the pollutants of paper mill effluent and their toxicity in the environment.  Conflict of interest The authors declare that there is no conflict of interest regarding the publication of this manuscript. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. Acknowledgement This study was funded by NFPwD F. No. 01-01/2019-Sch. to Ajay Kumar Singh Ph.D. scholar; University Grant Commission, New Delhi, to Adarsh Kumar Ph.D. scholar and the financial assistance from DBT, New Delhi, India Letter No. BT/PR18896/BCE/8/1372/2016 dated 28-03-2018 to Prof. Ram Chandra, is highly acknowledged.