Formation of resistance in the Chironomus plumosus to four pesticides over 45 generations

A study was conducted on the Chironomus plumosus larvae to determine initiation of resistance to four pesticides – chlorfenvinphos, chlorpyrifos, chlorpyrifos-methyl, and malathion. First generational LC₅₀ values were well within the threshold value for chironomids based upon the literature. Subsequent LC₅₀ values were observed to increase, indicating a lessening of the toxicity of the pesticides to the chironomid. In the case of chlorfenvinphos, the 96 h LC₅₀ for generations 1–23 was 6 µg L^?1, in generations 3–7 was 8.57 µg L^?1 and 11.14 µg L^?1 for generations 8–9. Generations 10–12 had an LC₅₀ value of 22.58 µg L^?1 and generation 13 had an LC₅₀ value of 35.08 µg L^?1. Generation 14 had an LC₅₀ value of 47.58 µg L^?1. Generations 15–19 and 20–24 had 96 h LC₅₀ values of 60.68 µg L^?1, 72.58 µg L^?1, 85.08 µg L^?1, 97.58 µg L^?1 and 110.08 µg L^?1, respectively. Generations 25, 26 to 30, 31 to 38 and 39 to 45 had 96 h LC₅₀ values of 160.42 µg L^?1, 210.7 µg L^?1, 262.24 µg L^?1 and 274.36 µg L^?1, respectively. The variation between LC₅₀ values was found to be statistically significant. This was observed for most pesticides tested. Larval size and life cycle duration was observed to change from generation to generation with the body size decreasing markedly from 1 to 0.3 cm with life cycle increasing from 7 to 39 days.     

Acute toxicity of ammonia to a freshwater teleost,Labeo bata larvae

Ammonia toxicity tests were performed with Labeo bata (bata) larvae of three different size groups. One hundred percent survival of larvae (500.0?±?4.0?mg) was recorded when exposed to ammonia concentrations of 1.0–13.56?mg?L^?1 at 96?h of exposure. Bata larvae exposed to ammonia concentrations of 15.8–25?mg?L^?1 showed 10–74% mortalities. The 96?h LC₅₀ value for 200 (±5), 250 (±2) and 500 (±4) mg bata larvae were 11.5, 16.8 and 22.5?mg?L^?1 un-ionised ammonia concentrations, respectively. When fish were exposed to different doses of ammonia, behavioural changes immediately occurred even at the lowest dose. At first, the fish became hyperexcitable, the skin darkened and they showed an increased ventilation frequency, fish behaviour became normal, 24?h after exposure. A 96?h LC₅₀ value of un-ionised ammonia showed direct relationship with the increasing size of bata larvae.     

A comparison of two methods to observe the effects of dual metal exposure to the chironomus larvae

A study was conducted to investigate the effects of metal mixtures on the Chironomus plumosus. Two methods of preparing the mixtures were used, the Toxic Unit (TU) method and the parts per million (PPM) + PPM method. A comparison was undertaken between the two methods to observe the ease with which (1) experiment could be conducted and (2) whether LC₅₀ values could be obtained. In this study, it was noted that the PPM + PPM method was an easier method to conduct metal mixture experiments as the LC₅₀ values could be easily calculated for individual metals and compared with the LC₅₀ values of metals obtained from single metal experiments. The findings showed that the LC₅₀ value for zinc (Zn) was more toxic for chromium (Cr) when it was reduced from 9.6 to 0.3 mg L^?1, with silver the LC₅₀ value became 3.6 mg L^?1, 4.5 mg L^?1 with nickel (Ni). In the PPM + PPM method, the LC₅₀ value for Zn was observed to be 10.2 mg L^?1 when in mixture with chromium, 23.8 mg L^?1 with silver, and 13.4 mg L^?1 with Ni. Changes in the 96 h LC₅₀ value were found to be significant for all metals.     

Endosulfan reduces fertilization success and causes abnormal embryo development to zebrafish

Abstract

Reproductive and teratogenic effects of endosulfan were investigated by exposure of mature breeding zebrafish (Danio rerio) to environmentally relevant concentrations of 0.01–1.2?μg L^?1 and monitoring their spawning, fertilization success, and gonad histology. Fertilization success was negatively correlated to duration of exposure at endosulfan concentration of 1.2?μg L^?1. No apparent effect was observed on fish gonads after histological examination. Teratogenic effects were assayed by exposing newly spawned embryos to endosulfan at 1.0–200?μg L^?1 and determining coagulation, tail development, eye formation, and spontaneous movements 24?h after fertilization; pigmentation, heart rate, edema, and mortality after 48?h and hatch time, mortality, and spinal deformations after 144?h. At 50–100?μg L^?1, endosulfan caused impaired tail development and differentiation, and at 10–200?μg L^?1 spinal deformation and increased mortality, depending upon the timing of exposure.     

4种甲氧基丙烯酸酯类杀菌剂不同剂型对斑马鱼急性毒性效应

采用半静态法测定了4种甲氧基丙烯酸酯类杀菌剂原药及制剂对斑马鱼(Brachydonio rerio)急性毒性。结果表明,以实测浓度计,250 g·L~(-1)嘧菌酯悬浮剂对斑马鱼的96 h半数致死浓度(LC50)值为0.539 mg·L~(-1),急性毒性为高毒,95%嘧菌酯原药和50%嘧菌酯水分散粒剂对斑马鱼的LC50(96 h)值分别为1.09和1.21 mg·L~(-1),急性毒性均为中毒;98%啶氧菌酯原药和22.5%的啶氧菌酯悬浮剂对斑马鱼的LC50(96 h)值分别为0.0974和0.0972 mg·L~(-1),急性毒性均为剧毒;95%吡唑醚菌酯原药、15%吡唑醚菌酯悬浮剂和250 g·L~(-1)吡唑醚菌酯乳油对斑马鱼的LC50(96 h)值为0.0613、0.0549和0.0487 mg·L~(-1),急性毒性均为剧毒;95%醚菌酯原药和50%醚菌酯水分散粒剂对斑马鱼的LC50(96 h)值分别为0.468和0.702 mg·L~(-1),急性毒性均为高毒。这4种甲氧基丙烯酸酯类杀菌剂对斑马鱼均有较高风险,会对其他水生生物也存在潜在的风险。     

Cardioprotective effect of Lagenaria siceraria (Mol) ameliorates isoproterenol-induced cardiac toxicity in rats by stabilizing cardiac mitochondrial and lysosomal enzymes

A study was conducted to determine the effect of the chironomid larval tube on heavy metal toxicity. Silica gel was provided as a substrate with which the chironomid larvae could construct larval tubes. A series of 96 h LC₅₀ bioassays were conducted with and without the presence of the silica gel substrate, to allow comparison between the two test conditions. From the study it was observed that a 10–15% variation in LC₅₀ value occurred. The 96 h LC₅₀ for copper at the temperatures 35, 28, 20, 15, and 10°C was observed to be 7.5, 9.5, 7.5, 4.5, 3.6, and 2.8 mg L^?1, respectively. The same test when conducted in the presence of silica gel produced LC₅₀ values of 26.50, 33.50, 15.82, 12.77, and 9.94 mg L^?1 for the same temperatures. This would demonstrate that the larval tube did, to some extent, protect the larvae from the effects of the heavy metal.     

Acute toxicity of benzophenone-type UV filters and paraben preservatives to freshwater planarian,Dugesia japonica

Fourteen benzophenone-type UV filters and four paraben preservatives were selected to examine their acute toxicities on Dugesia japonica. The 48-h LC₅₀ values for planarians exposed to benzophenone-type UV filters can be ranked as oxybenzone?>?mexenone?>?5-chloro-2-hydroxybenzophenone?> 2,4-dihydroxybenzophenone?>?2-hydroxybenzophenone?>?dioxybenzone?>?benzophenone?>?2,2′,4,4′-tetrahydroxybenzophenone?>?4-hydroxybenzophenone?> 3-hydroxybenzophenone?>?4,4′-dihydroxybenzophenone?>?2,2′-dihydroxy-4,4′-dimethoxybenzophenone?>?2,3,4-trihydroxybenzophenone?>?sulisobenzone with a range from 0.9 to 145?mg?L^?1 with a similar sequence for the 96?h LC₅₀ values, ranging from 0.5 to 77?mg?L^?1. The 48 and 96?LC₅₀ values for planarians exposed to paraben preservatives can be ranked as butylparaben?>?propylparaben?>?ethylparaben?>?methylparaben. Among all the tested chemicals, oxybenzone was the most toxic and sulisobenzone the least toxic chemical to planarian at each exposure period. Most benzophenone-type UV filters are toxic to aquatic animals with 48?h LC₅₀ values less than 10 mg?L^?1, except for 2,2′-dihydroxy-4,4′-dimethoxybenzophenone, 2,3,4-trihydroxybenzophenone, and sulisobenzone. Because of their common occurrence in aquatic environment, more studies on aquatic toxicities of benzophenone-type UV filters and paraben preservativs are needed to provide important information to adequately assess their ecological risk.     

Deltamethrin-induced alterations in serum calcium and prolactin cells of a freshwater teleost,Heteropneustes fossilis

Deltamethrin, a pyrethroid insecticide widely used in many countries, causes significant adverse effects in aquatic ecosystems. The concentration of deltamethrin in water reservoirs and the run off from agricultural areas (in water) in many countries range up to 24.0?µg?L^?1 which is higher than the recommendation of the European Union standard. Hence, in this study the effects of deltamethrin were investigated, i.e. its toxic impacts on the freshwater catfish Heteropneustes fossilis in terms of serum calcium and prolactin cells (located in the rostral pars distalis region of the pituitary). The fish were subjected to deltamethrin for a short-term experiment (96?h; 1.5?µg?L^?1 e.0.8 of 96?h LC₅₀) and a long-term experiment (28 days; 0.37?µg?L^?1 e.0.2 of 96?h LC₅₀). After short-term deltamethrin exposure, serum calcium levels decrease. No histological change in prolactin cells is noticed throughout the short-term experiment. Fish exposed to deltamethrin for 7 days also exhibit a decrease in serum calcium level. This decrease persisted until the end of the experiment (28 days). Prolactin cells of fish treated for 14 days with deltamethrin exhibit increased nuclear volume and degranulation, increasing progressively from the 21st day onwards. After 28 days, a few degenerated cells are discerned. The results of this study show that deltamethrin is moderately toxic for the freshwater fish H. fossilis by producing adverse effects on serum calcium and prolactin cells. Hence, it should be used with caution in areas near fish-inhabited waters.     

Uptake and toxicity of some pesticides on three freshwater fishes (Oreochromis niloticus,Clarias gariepinus and Chrysicthys nigrodigitatus)

Studies were carried out to determine the toxicity of some selected pesticides on fresh water fish in a tropical environment. The uptake of the pesticides lindane, pentachlorophenol (PCP), and propoxur, which are frequently used by farmers and industrialists were studied in concrete ponds at the University of Cape Coast, in Ghana. The fishes used for the study were Oreochromis niloticus, Clarias gariepinus, and Chrysicthys nigrodigitatus, and were obtained from cultured ponds in the Cape Coast District and Mankessim in the Central Region and Weija Dam, in the Greater Accra region of Ghana. Single high lethal concentration (SD) or acute treatment and cumulative/chronic (or multiple minor) lethal concentration (CD) treatment were employed in administering the pesticides to the fishes via water. Gas chromatograph (GC) Electron Capture Detector (ECD) analysis was done on the dead fishes to see the extent of ingestion. The LC₅₀ values obtained for lindane on the three fish samples were as follows: Chrysicthys?–?0.38?mg?L^?1; Oreochromis?–?0.42?mg?L^?1, and Clarias?–?1.2?mg?L^?1. Mortalities occurred in fish within 3–5 days of application. For the PCP on Chrysicthys, Oreochromis, and Clarias species the LC₅₀ values were 0.42, 0.32 and 0.64?mg?L^?1, respectively for over a 2–3 day period. For a three-time influx period of propoxur the LC₅₀ for Chrysicthys, Oreochromis, and Clarias, were 22.0, 30.40, and 45.04 (all in mg?L^?1), respectively. The results obtained indicated that the pesticides had adverse effects on the general growth and reproduction of fishes. Gonadosomatic indices also showed that the pesticides affected the development of the body, the gonads, and their reproduction.     

Larvicidal potential of different solvent extracts and oleanolic acid derivative from Coccinia indica against vector mosquitoes

The mosquito larvicidal activities of different solvent extracts (chloroform, ethyl acetate, and methanol) from the leaves of Coccinia indica were studied. Among the extracts tested, the methanol extract showed the highest larvicidal activity against early fourth instar larvae of Culex quinquefasciatus (LC₅₀?=?142?mg?L^?1), Aedes aegypti (LC₅₀?=?158?mg?L^?1) and Anopheles stephensi (LC₅₀?=?161?mg?L^?1) after 24?h of exposure period. The lowest larval mortality was observed with the ethyl acetate extract with the LC₅₀ values of 927, 872, and 730?mg?L^?1 against Cx. quinquefasciatus, Ae. aegypti, and An. stephensi, respectively. The chloroform extract showed the moderate larvicidal activity against the mosquito species tested with the LC₅₀ values from 446 to 546?mg?L^?1. A new triterpene was isolated from the methanol extract using bio-assay guided separation and identified as an oleanolic acid derivative by NMR, IR and mass spectral studies. The compound showed prominent larvicidal activity against Cx. quinquefasciatus, Ae. aegypti and An. stephensi with the LC₅₀ values of 5.6, 5.0, and 4.8?mg?L^?1, respectively. The results of the present investigation offer an opportunity for developing alternatives to rather expensive and environmentally hazardous organic insecticides.     

Responses of the Indian major carp Labeo rohita to deltamethrin at acute and sublethal concentrations

The effects of deltamethrin at acute and sublethal concentrations on some hematological and biochemical profiles in the Indian major carp Labeo rohita have been examined. The lethal concentrations (LC₅₀) of deltamethrin for 24 and 96 h were found to be 0.44 and 0.38 mg L^?1, respectively. During acute treatment (24 h), hemoglobin (Hb), hematocrit (Hct), red blood cell (RBC), white blood cell (WBC), mean cellular volume (MCV), mean cellular hemoglobin (MCH), and mean cellular hemoglobin concentration (MCHC) values were significantly increased compared to control group. Plasma glucose, protein, cholesterol, albumin, globulin, triglyceride, and glycogen in gill and muscle were significantly decreased. Upon sublethal treatment (0.044 mg L^?1, 10th of 24 h LC₅₀), RBC and the biochemical parameters, except glucose and cholesterol, decreased significantly, while MCV, MCH, and MCHC values increased up to the 28th day and then declined. Cholesterol level was significantly increased throughout the study period. A biphasic response in the levels of Hb, Hct, WBC, and glucose were observed during sublethal treatment.     

Alterations in serum electrolyte levels after dimethoate exposure and recovery in the freshwater air-breathing catfish,Heteropneustes fossilis (Bloch)

Dimethoate, a moderately toxic insecticide, has a wide range of agricultural and domestic applications. Like other organophosphates, dimethoate has anticholinesterase activity. Fish are non-target organisms, inadvertently exposed to pesticides and their metabolic products. The present study includes short-term (96 h) and long-term (36 d) effects of dimethoate exposure on some serum electrolytes Ca²⁺, Mg²⁺, and Pi in the freshwater air-breathing catfish Heteropneustes fossilis. The concentration of dimethoate for short-term exposure was 2.24 mg L^?1 (75% of 96 h LC₅₀) and for long-term exposure 0.75 mg L^?1 (25% of 96 h LC₅₀). The study includes the recovery pattern in serum electrolytes after placing the fish in pesticide-free water. Fish show hypocalcemia, hypermagnesemia, and hyperphosphatemia after short-term and long-term exposure to dimethoate. When placed in pesticide-free water, these electrolytes exhibit recovery towards normalization, indicating significant (p < 0.05) recovery.     

Toxicity of metals to tadpoles of the common Sunda toad,Duttaphrynus melanostictus

Tadpoles of the common freshwater Sunda toad, Duttaphrynus melanostictus (Amphibia, Bufonidae), were exposed for a 4-day period under laboratory conditions to copper (Cu), cadmium (Cd), zinc (Zn), lead (Pb), nickel (Ni), iron (Fe), aluminum (Al), and manganese (Mn) at various concentrations. Mortality was assessed and median times of death (LT₅₀) and lethal concentrations (LC₅₀) were calculated. LT₅₀ and LC₅₀ increased with the decrease in mean exposure times and concentrations for all metals. LC₅₀ (96?h) for Cu, Cd, Zn, Pb, Ni, Fe, Al, and Mn were 0.03, 0.3, 4.2, 1.5, 8.8, 0.4, 1.9, and 39?mg?L^?1, respectively. Cu was the most toxic to D. melanostictus, followed by Cd, Fe, Al, Pb, Zn, Ni, and Mn (Cu?>?Cd?>?Fe?>?Al?>?Pb?>?Zn?>?Ni?>?Mn). Duttaphrynus melanostictus is similarly sensitive to these metals as other amphibian tadpoles.     

丙硫菌唑对斑马鱼的安全性评价及其生物富集行为研究

丙硫菌唑是一种市场前景非常好的新型广谱杀菌剂。本文研究了丙硫菌唑对水生生物斑马鱼的急性毒性和生物累积风险。通过斑马鱼的急性毒性试验获得丙硫菌唑对斑马鱼的96 h-LC₅₀为2.06 mg a.i. L^-1。随后,采用0.02 mg L^-1 (1/100LC₅₀)和0.2 mg L^-1 (1/10LC₅₀)2个浓度的丙硫菌唑,通过8 d实验,获得其在斑马鱼体内的生物累积效应。在0.02 mg L^-1组中,第8天时,斑马鱼体内的浓度达到0.733 mg kg^-1,生物富集系数(BCF_{8 d})缓慢增长到34.36。而在0.2 mg L^-1组,第8天时,斑马鱼组织内丙硫菌唑浓度为4.198 mg kg^-1, BCF_{8 d}值为19.72。结果表明,丙硫菌唑对斑马鱼的毒性等级为中毒,同时其在斑马鱼体内具有中等生物累积效应。因此,在使用过程中,需要考虑其对水生生物的毒性和水生食物链的放大效应。     

Acute and chronic toxic effect of lead (Pb) and zinc (Zn) on biomarker response in post larvae of Penaeus monodon (Fabricus, 1798)

Metal pollution produces damage to marine organisms at the cellular level possibly leading to ecological imbalance. The present investigation focused on the acute and chronic toxicity of lead (Pb) and zinc (Zn) by examining the effects of biomarker enzymes in post-larvae of Penaeus monodon (Tiger prawn). Antioxidant biomarker responses such as lipid peroxidation (LPO) and catalase (CAT) activity for Pb and Zn were determined following chronic exposure. Acute Lethal Concentration ₅₀ (LC₅₀) values observed in the study at 96?h for Pb and Zn at 5.77?±?0.32?mg?L^?1 and 3.02?±?0.82?mg?L^?1, respectively. The estimated No Observed Effect Concentration and Lowest Observed Effect Concentration values for Pb were 0.014 and 0.029?mg?L^?1 and that recorded for Zn was 0.011 and 0.022?mg?L^?1, respectively. Among the two metals studied, toxicity of Zn was found to be greater to P. monodon than Pb. The activities of antioxidant defense enzymes and total protein content differed significantly from control following exposure to both metals. Overall, the biomarker studies demonstrated that alterations in antioxidant enzymes and induction of LPO reflect the consequences of heavy metal exposure in P. monodon.     

Quantal response of Paleomonetes africanus in locally formulated drilling mud lubricants

The toxic potency of three locally formulated drilling mud lubricants against Paleomonetes africanus, a brackish water crustacean commonly found in the oil producing Niger Delta (Nigeria), was evaluated. A 96?h exposure to quantal (lethal) doses of the mud lubricants gave LC₅₀ (median lethal concentration) of 350, 620 and 210?mg?L^?1, respectively. The mud lubricant with the highest molecular weight of 40,000?g?mol^?1 (lubricant 2) had the least toxicity on the organisms, attributable to the reduced rate of diffusion into the vital cells of the test organism. The higher toxicity of lubricant 3 over lubricant 1, despite its higher molecular weight, might be as a result of its higher acid value of 21.60?mg?KOH?g^?1. The results show that the test lubricants would be environmentally friendly within the baseline concentrations and lethal times (LT₅₀) measured.     

The influence of solid matrices on arsenic toxicity to Corophium orientale

In marine ecosystems, benthic organisms are really important because they are the first step in the transfer of contaminants from environment to biota. To this end, this study focused on biological assays with the amphipod Corophium orientale exposed to two different molecules of arsenic: arsenate (As^V), the most abundant form in sediments, and dimethyl-arsinate (DMA), expected to be moderately toxic as an intermediate in the process of detoxification. The toxicity of arsenic compounds was measured after exposure to three different matrices: water, spiked natural sediment and inert spiked quartz sand. LC₅₀ values were calculated for each exposure, and the results confirmed the highest toxicity of As^V, in addition to underlining the importance of matrix of exposure. Water exposure was the matrix which presented the highest toxicity for inorganic arsenic (As^V LC₅₀=3.51 mg L^?1 vs DMA LC₅₀=54.65 mg L^?1), spiked natural sediment demonstrated its capability to chelate arsenate toxicity (As^V LC₅₀=34.27 mg kg^?1 vs. DMA LC₅₀=52.19 mg kg^?1) and spiked quartz sand presented intermediate values for As^V (LC₅₀=25.26 mg kg^?1), whereas for DMA a lower toxicity was registered (LC₅₀=872.35 mg kg^?1). This study can provide some useful data linked with chemical speciation of arsenic and exposure matrix, for improving the correct management of contaminated sediment.     

Acute toxicity of 30 pharmaceutically active compounds to freshwater planarians,Dugesia japonica

Pharmaceutically active compounds are produced worldwide and consumed in large quantities, so these chemicals are frequently detected in limnic environments posing potential ecological risks. Thirty pharmaceutically active compounds were selected for examination of their acute toxicity for freshwater planarians (Dugesia japonica). Among the 30 compounds tested, diclofenac, mefenamic acid, naproxen, propranolol HCl, and diphenhydramine HCl had a 48-h nominal LC₅₀ below10 mg L^?1, and for 18 chemicals, it exceeded 100 mg L^?1. The 96-h nominal LC₅₀ was below 10 mg L^?1 for diclofenac, mefenamic acid, naproxen, propranolol HCl, diltiazem HCl, diphenhydramine HCl, hydroxyzine HCl, and triprolidine HCl, and for 15 chemicals, it exceeded 100 mg L^?1. Among different therapeutic groups, analgesics were most toxic to planarians, and antibiotics were least toxic. Antihistamines and beta blockers varied in their acute toxicity to planarians. At the current environmental levels, none of the tested pharmaceutically active compounds may have acutely harmful impacts on aquatic invertebrates. To answer the question whether chronic, long-term exposure to pharmaceutically active compounds may entail ecological risks for aquatic ecosystems, further investigations with different end points in multiple species tests are needed.     

Acute toxicity of waterborne manganese to Rutilus caspicus (Yakovlev, 1870) – gill histopathology,immune indices,oxidative condition,and saltwater resistance

The acute toxicity of manganese (Mn) in Caspian roach Rutilus caspicus was determined. The 96 h-LC₅₀ was calculated to be 300 (240–360) mg L^?1. Then, fish were exposed for 96 h to 300 (96 h-LC₅₀), 150 (50% of 96 h-LC₅₀), 60 (20% of 96 h-LC₅₀), and 0 (control group) mg L^?1 Mn. Exposure to all Mn concentrations led to fish gill damages, including epithelial lifting, hyperplasia, lamellar fusion, aneurism, and lamellar curling. Also, the fish exposed to all Mn concentrations showed decreased plasma alternative complement and lysozyme activity. Whole body superoxide dismutase, catalase, and malondialdehyde showed significant increases in the Mn-exposed fish which also exhibited lower tolerance to saltwater than the control group.     

Assessment of toxicity of two nitroaromatic compounds in the freshwater fish Cyprinus carpio

This study was conducted to evaluate the toxicological response of p-nitrotoluene and p-nitroaniline to the key fish species, Cyprinus carpio. A freshwater fish bioassay based on the 96 h LC ₅₀ was used to estimate the single and joint toxicity of the two chemicals. The toxicity of p-nitrotoluene was greater than that of p-nitroaniline based on 96 h LC ₅₀ values of 40.74 mg·L^?1 and 48.99 mg·L^?1, respectively. Both compounds had moderate toxicity toward Cyprinus carpio, and this toxicity increased with the exposure duration and concentration. Binary mixtures of the compounds were more toxic than the individual compounds at 96 h, and they acted upon partial addition. When the exposure time was longer, the toxicity increased for mixtures of compounds with the same concentration or toxicity. The results of this study suggest that exposure to a combination of these chemicals would result in a higher environmental risk in aquatic systems than exposure to either compound alone. Further research is needed to investigate the combined effects and sublethal toxicity of p-nitrotoluene and p-nitroaniline, since they are both still used in China.