Phage composition and application thereof in strengthening degradation of nitrochlorobenzene in soil

Abstract

The invention relates to a bacteriophage composition and an application thereof in enhancing nitrochlorobenzene degradation in soil, the bacteriophage composition comprises three bacteriophages which are klebsiella pneumoniae bacteriophage phi YSZKA with the preservation number of CCTCC NO: M 2018513, klebsiella pneumoniae bacteriophage phi YSZKB with the preservation number of CCTCC NO: M 2018513, the invention discloses a multivalent phage phi YSZPK for attacking klebsiella pneumoniae and pseudomonas aeruginosa, and the preservation number of the multivalent phage phi YSZPK is CCTCC (China Center For Type Culture Collection) NO: M 2018516. The preservation number of the bacillus cereus phage phi YSZBA1 is CCTCC (China Center For Type Culture Collection) NO: M 2018517. The transplanted bacteriophage can promote nitrochlorobenzene degradation metabolism of host bacteria by adjusting a microbial community structure and providing an auxiliary metabolism gene mode. The invention provides a green and sustainable microorganism synergistic degradation technology, and provides an effective scheme for degradation treatment of harmful chemical substances in specific soil types in China.

Description

Technical Field

[0001] The present invention belongs to the technical field of phages, and specifically relates to the application of soil phage-host combined degradation of pesticides, aiming to enhance the degradation efficiency of nitrochlorobenzene in soil by optimizing the soil microbial community structure. Background Art

[0002] Nitrochlorobenzene (NCBs) is a typical intermediate compound of organochlorine pesticides, with significant "carcinogenic, teratogenic, and mutagenic" toxic effects. The soil polluted by NCBs is characterized by wide area, heavy pollution, and great difficulty in treatment, seriously threatening human health and environmental safety, and is a soil pollution problem that urgently needs to be solved. As the "first life battlefield" to positively respond to the stress of NCBs pollution, the soil microbiome plays important ecological and environmental functions such as toxicity indication, functional strain resource reserve, and combined detoxification and remediation. Therefore, the development of new pesticide degradation technologies, especially the enhancement of the degradation efficiency of toxic pollutants by improving the microbial community, has become an important research direction in current environmental governance.

[0003] Due to the complexity of the soil environment, a single microbial community is often difficult to play a role in the degradation of NCBs on a large scale. Therefore, exploring and utilizing the cross-kingdom interactions between microorganisms has become one of the key technologies to improve the degradation efficiency of soil pollutants. Bacterial phages (referred to as phages for short) and host bacteria are typical examples of cross-kingdom interactions. Phages are organisms that specifically prey on living host bacteria and survive, and are widely distributed in soil, air, and water. It is estimated that their total amount reaches 10 31 orders of magnitude. Phages can compensate for the expression of various physiological functions of the host through horizontal gene transfer of auxiliary metabolic genes (AMGs), promoting the host bacteria to efficiently degrade harmful substances, and having the potential for environmental remediation.

[0004] Phage transplantation refers to an emerging biological technology that efficiently enriches the overall phage community in a habitat, and then transplants it into a designated target host environment and enables it to colonize and play a role. Soil phage transplantation is derived from the concept of faecal microbiota transplantation in the medical field for the treatment of human diseases. The advantages of soil phage transplantation technology are: it can quantitatively identify the roles and functions of phages in the soil microbiome, and facilitate the transplantation of known lysogenic phages carrying NCBs-degrading functional AMGs into the target soil to successfully assist the host bacteria in strengthening their own pesticide degradation metabolic pathways.

[0005] Through the review and analysis of relevant literature and patents, no technical solution has been found to apply phage transplantation technology to optimize the soil microbial system and enhance the degradation of nitrochlorobenzene. In the existing technology, the technology closest to the present invention is the soil pollution remediation technology based on microbial agents. For example, the invention publication number CN117778241A uses a strain of Agrobacterium tumefaciens to degrade 4-nitrochlorobenzene; while the invention publication numbers CN114196407A, CN119351117A, and CN108356070B are the application of mixed agents such as chemical oxidation remediation agents (nano-carbon, Fenton reagent, pH regulator) and soil remediation materials contaminated with organic pollutants (bamboo charcoal, graphene oxide, soybean straw, biochar, and enzyme solution), and strengthen the soil nitrochlorobenzene pollution from the physical and chemical levels by ensuring the stability of the reaction system. However, the above inventions do not involve microbial cross-interactions, and there is still a general lack of application of the phage-host co-degradation metabolic pathway widely existing in the soil.

[0006] The main defects of the existing technology are as follows: The existing soil pollution remediation technologies mainly rely on physical, chemical, and microbial methods, but there are various defects: 1) The chemical method has low degradation efficiency and may have a negative impact on the ecological environment; 2) The microbial agents are single, and the remediation is restricted by the soil environment and pollutant types, and the degradation effect is unstable; 3) Exogenous microorganisms are easily affected by competition in the soil, with poor stability and a decline in degradation efficiency; 4) Traditional microbial remediation has a risk of secondary biological pollution, and is costly and time-consuming. Therefore, there is an urgent need for an efficient, stable, and low-risk remediation technology to improve the pollutant degradation efficiency and reduce the ecological impact.

[0007] The main reasons for the defects are as follows: The existing remediation technologies have not fully considered the complexity and dynamics of the soil microbial community. Traditional microbial remediation methods mostly rely on the inoculation of exogenous microorganisms, which are easily affected by the competition and ecological niche exclusion of indigenous flora, resulting in the inability of exogenous microorganisms to play a long-term and stable role. In addition, chemical remediation methods have high environmental pollution risks and costs, and cannot accurately degrade complex pollutant types. The existing technology lacks an innovative method that can improve the remediation efficiency, reduce the ecological risk, and enhance the remediation stability. Summary of the Invention

[0008] Technical problems to be solved: In view of the above-mentioned defects of the existing technology, the invention provides a phage composition and its application in enhancing the degradation of soil nitrochlorobenzene. This technical solution uses a phage suspension with the function of degrading NCBs after separation and purification, injects it into the target soil, and strengthens the soil pesticide degradation metabolic pathway by the cooperation of phage-injected AMGs and host bacteria. At the same time, the phage has the function of regulating the soil microbial community from bottom to top, further optimizing the indigenous bacterial symbiotic network, and improving the soil NCBs digestion potential.

[0009] Technical solution: A phage composition includes three strains of phages, all of which were deposited at the China Center for Type Culture Collection on August 1, 2018. They are Klebsiella pneumoniae phage The deposit number is: CCTCC NO:M 2018513, and the taxonomic name is Klebsiella phage A multivalent phage that attacks Klebsiella pneumoniae and Pseudomonas aeruginosa The deposit number is: CCTCC NO:M 2018516, and the taxonomic name is Pseudomonas aeruginosa and Klebsiella phage Bacillus cereus phage The deposit number is: CCTCC NO:M 2018517, and the taxonomic name is Bacillus cereus phage

[0010] Preferably, the mass ratio of the addition of the above three strains of phages is 1:1:1.

[0011] The above phage composition also contains a stabilizer, and the stabilizer is selected from at least one of trehalose, glycerol, gelatin or bovine serum albumin, and is used to improve the stability of the phage during storage and use.

[0012] The above phage composition contains at least one carrier, and the carrier is selected from at least one of sterile water, physiological saline, buffer solution or biocompatible polymer solution, and is used to facilitate the application and storage of the phage.

[0013] Application of the above phage composition in enhancing the degradation of nitrochlorobenzene in soil.

[0014] The above soil is soil for growing wheat, lettuce or carrots.

[0015] The ratio of mixing the above phage composition with the soil is to add 10 7 -10 8 PFU / g of the phage composition.

[0016] An agent for enhancing the degradation of nitrochlorobenzene in soil, and the active ingredient of the agent is the above phage composition.

[0017] The above agent for enhancing the degradation of nitrochlorobenzene in soil also includes auxiliary components, and the auxiliary components are selected from at least one of organic fertilizers, microbial inoculants, plant growth regulators or soil conditioners, and are used to synergistically enhance soil fertility and the nitrochlorobenzene degradation effect.

[0018] The working principle of the present invention is as follows: 1. Bacteriophages are a type of bacterial virus composed of a protein capsid (60%) and nucleic acid (40%), without a complete mature cell structure, and can be divided into lytic and lysogenic types; 2. Lysogenic bacteriophages can inject their own genetic material into bacteria, affecting the metabolic processes of the host bacterial community. Auxiliary metabolic genes (AMGs) are a segment of gene fragments present in the bacteriophage genome, generally randomly packaged and assembled into the bacteriophage genome during the synthesis of bacteriophage particles. Therefore, AMGs can affect the degradation process of pollutants (such as nitrochlorobenzene) by the host; 3. Broad-spectrum bacteriophages refer to bacteriophages that can infect two or more host bacteria with similar homology or between different species and genera, which is conducive to widely exerting the synergistic metabolic potential of bacteriophages; 4. The bacteriophages selected for the bacteriophage transplantation technology are based on the natural (temperature, moisture, pH) and non-natural (heavy metals, pesticides, antibiotics) environmental conditions of the in-situ soil. The bacteriophages selected for this technology are broad-spectrum lysogenic bacteriophages with the function of degrading NCBs. This bacteriophage can better integrate into the local microbial network, achieving a better bacteriophage-host synergistic pesticide degradation effect while minimizing ecological risks, ensuring the diversity and stability of the local microbial ecological functions; 5. The length of bacteriophages is about 20-200 μm, which is hundreds and thousands of times smaller than bacteria. Therefore, it can achieve wide migration in the soil, which is conducive to saving the application cost.

[0019] Beneficial effects: 1. The bacteriophage composition can promote the pollutant degradation function of host bacteria through the injection of AMGs, effectively improving the degradation efficiency of nitrochlorobenzene in the soil; 2. Lysogenic bacteriophages weaken the interspecies competition problem existing in traditional pollutant degradation agents, optimizing the microbial community structure from the perspective of cross-border cooperation; 3. The materials for preparing the buffer solution are easily available, convenient for storage, transportation, and operation, and can effectively control costs. Description of the Drawings

[0020] Figure 1 It is a schematic diagram of the application technology of bacteriophage transplantation in promoting soil carbon sequestration.

[0021] Figure 2 is a transmission electron micrograph of a bacteriophage

[0022] Figure 3 is a transmission electron micrograph of a bacteriophage

[0023] Figure 4 is a transmission electron micrograph of a bacteriophage

[0024] Figure 5 ​​​It is the abundance of the nitrochlorobenzene-degrading gene L-DEX 70 days after the injection of the microbial agent into the soil of the vegetable base.

[0025] Figure 6 It is the degradation rate of nitrochlorobenzene in the soil 70 days after the injection of the microbial agent into the soil of the vegetable base.

[0026] Figure 7 It is the abundance of the nitrochlorobenzene-degrading gene L-DEX 70 days after the injection of the microbial agent into the soil at Fengqiu Station in Henan Province.

[0027] Figure 8 It is the degradation rate of nitrochlorobenzene in the soil 70 days after the injection of the microbial agent into the soil at Fengqiu Station in Henan Province.

[0028] Figure 9 It is the abundance of the nitrochlorobenzene-degrading gene L-DEX 60 days after the injection of the microbial agent into the soil of a relocated site contaminated with a certain fungicide pesticide.

[0029] Figure 10 It is the degradation rate of nitrochlorobenzene in the soil 60 days after the injection of the microbial agent into the soil of a relocated site contaminated with a certain fungicide pesticide. Specific implementation manners

[0030] The following specific implementation manners do not limit the technical solution of the present invention in any form. Any technical solution obtained by means of equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

[0031] Table 1 Determination of the optimal multiplicity of infection

[0032]

[0033] Table 2 Determination of the optimal multiplicity of infection

[0034]

[0035] Table 3 Determination of the optimal multiplicity of infection

[0036]

[0037] Klebsiella pneumoniae phage The preservation number is: CCTCC NO: M 2018513, and the taxonomic name is Klebsiella phage Preserved in Wuhan University, Wuhan, China;

[0038] Multivalent phage attacking Klebsiella pneumoniae and Pseudomonas aeruginosa The preservation number is: CCTCC NO: M2018516, and the taxonomic name is Pseudomonas aeruginosa and Klebsiella phage It is preserved in Wuhan University, Wuhan, China;

[0039] Bacillus cereus phage The preservation number is: CCTCC NO: M 2018517, and the taxonomic name is Bacillus cereus phage It is preserved in Wuhan University, Wuhan, China.

[0040] The nitrochlorobenzene degradation gene L-DEX refers to the nitrochlorobenzene degradation gene carried by phages detected in the soil.

[0041] Example 1:

[0042] The tested potting soil was collected from the soil of a vegetable base. Xinmai 26 was planted. The basic physical and chemical properties of the soil are as follows: sand particles 26.8%, silt particles 37.4%, clay particles 31.82%, pH 7.24, total nitrogen 1.44 g·kg -1 , water-soluble nitrogen 1.62 g·kg -1 , total phosphorus 1.50 g·kg -1 , total potassium 18.52 g·kg -1 , CEC 16.43 cmol·kg -1 .

[0043] Weigh out 0.5 g of soil and put it into a 15 mL sterile centrifuge tube. Add 4.5 mL of liquid medium to the sample, mix thoroughly and incubate for 1 h, then centrifuge. The supernatant obtained by centrifugation is filtered with a tangential flow filtration system. After tangential flow filtration, the filtrate is a phage suspension, which suspends free phages in the liquid component and is often inverted during incubation. Separate the filtered phages: add 100 μL of nitrochlorobenzene-degrading functional host bacteria (Klebsiella) in the logarithmic phase + 200 μL of phage suspension + 5 mL of semi-solid medium, pour it into solid LB medium, and incubate it upside down at 30-37 °C after solidification. Continue with phage purification: Pipette 100 μL of the dilution and 100 μL of bacteria to spread on a double-layer plate and purify continuously for 3-5 times until the phage plaques on the plate are of the same size. After cultivation, it can be seen that the phage plaques on the culture dish are transparent in the middle, have no halo around, and have a diameter of about 2-3 mm, and Klebsiella pneumoniae phage is obtained Pick a single clear and transparent phage plaque for enrichment, mix it with 50 wt.% glycerol at a volume ratio of 1:1, and store it at -80 °C for later use.

[0044] Klebsiella pneumoniae phage obtained based on the above-mentioned Klebsiella as the host bacterium Carry out the process of accelerating the expression of phages with broad host ranges: Take 600 μL of the preserved phage stock solution, and mix it with 200 μL of Klebsiella pneumoniae and 200 mL of Bacillus cereus mixed bacterial suspension. Then add them to 99 mL of LB liquid medium, and add solid calcium chloride to adjust the final concentration to 1 mmol·L -1 , incubate at 37 °C with shaking at 150 rpm for 96 h. Sampling is carried out every 8 h. The phages obtained by centrifugation and filtration are used to pour double-layer plates with Bacillus cereus for verification. Observe the plaques. If plaques appear, it proves that the directed evolution is successful, and a multivalent phage attacking Klebsiella pneumoniae and Pseudomonas aeruginosa is obtained and Bacillus cereus phage Pick a single clear and transparent plaque for enrichment, mix it with 50 wt.% glycerol in a volume ratio of 1:1, and store it at -80 °C for later use

[0045] Based on the above operations, three phages are obtained, namely: Klebsiella pneumoniae phage Multivalent phage attacking Klebsiella pneumoniae and Pseudomonas aeruginosa (broad-spectrum), Bacillus cereus phage

[0046] A total of five groups of treatments are set in the experiment: ① Control group (CK): One square meter of wheat (covering the seeds with 0.5 - 1 cm of soil, room temperature 25 ± 2 °C, applying 60 mg / kg of nitrochlorobenzene); ② Single phage treatment (V1): On the basis of the control group, inoculate 100 mL of a phage suspension with a concentration of 10 6 pfu·mL -1 of phage suspension; ③ Single phage treatment (V2): On the basis of the control group, inoculate 100 mL of a phage suspension with a concentration of 10 6 pfu·mL -1 of phage suspension; ④ Single phage treatment (V3): On the basis of the control group, inoculate 100 mL of a phage suspension with a concentration of 10 6 pfu·mL -1 of phage suspension; ⑤ Mixed phage treatment (V): On the basis of the control group, inoculate 100 mL of a mixed phage suspension of the above three phages with a concentration of 10 6 pfu·mL -1 The mass ratio of the three phages in the mixture is 1:1:1. The injection bit uses a direct-push injection tube with a diameter of 2 - 5 cm, which is suitable for agricultural soil; the injection depth is 20 - 40 cm soil layer; the positioning accuracy of the robotic arm is ≤ 2 mm to ensure the accuracy of multi-point repeated sampling. Soil is sampled on-site 70 days after the growth of chili peppers, and the abundances of the nitrochlorobenzene degradation gene L-DEX in the soil under the five treatments of CK, V1, V2, V3, and V are measured as: 4.72×105 copies·g -1 、6.24×10 7 copies·g -1 、5.49×10 8 copies·g -1 、3.86×10 8 copies·g -1 、9.23×10 8 copies·g -1 ( Figure 5 )。The abundance of the nitrochlorobenzene degradation gene L-DEX in the soil of the V treatment group increased by three orders of magnitude compared with that of the control group (p<0.05). The nitrochlorobenzene degradation rates in the treatment group and the control group were 3.21%, 18.13%, 10.36%, 9.78%, and 95.34% respectively( Figure 6 ). The above results indicate that the addition of the phage suspension has a significant effect on the degradation of nitrochlorobenzene in the soil.

[0047] Example 2:

[0048] Basic physical and chemical properties of the soil: pH 6.8, organic matter 21.1 g·kg -1 , total nitrogen 4.8 g·kg -1 , total phosphorus 2.1 g·kg -1 , the soil mechanical composition is 45.1% sand grains (sandy soil), 22.8% silt grains, and 32.1% clay grains.

[0049] A total of five groups of treatments were set up in the experiment: ① Control group (CK): 3 carrots were planted in each pot (covering the seeds with 0.5 - 1 cm of soil, room temperature 25±2°C, applying 60 mg / kg of nitrochlorobenzene); ② Single phage treatment (V1): On the basis of the control group, 100 mL of a phage suspension with a concentration of 10 6 pfu·mL -1 was inoculated; ③ Single phage treatment (V2): On the basis of the control group, 100 mL of a phage suspension with a concentration of 10 was inoculated; ④ Single phage treatment (V3): On the basis of the control group, 100 mL of a phage suspension with a concentration of 10 6 pfu·mL -1 was inoculated; ⑤ Mixed phage treatment (V): On the basis of the control group, 100 mL of a phage suspension with a concentration of 10 was inoculated; ⑤ Mixed phage treatment (V): On the basis of the control group, 100 mL of a phage suspension with a concentration of 10 6 pfu·mL -1 was inoculated; ⑤ Mixed phage treatment (V): On the basis of the control group, 100 mL of a phage suspension with a concentration of 10 was inoculated; ⑤ Mixed phage treatment (V): On the basis of the control group, 100 mL of a phage suspension with a concentration of 10 6 pfu·mL -1The above-mentioned mixed suspension of three phages, with a mass ratio of the three phages in the mixture of 1:1:1, uses a direct-push injection tube with a diameter of 2 - 5 cm for the injection bit, which is applicable to agricultural soil; the injection depth is in the soil layer of 20 - 40 cm; the positioning accuracy of the robotic arm is ≤ 2 mm to ensure the accuracy of multi-point repeated sample application. Soil was sampled on-site 70 days after the growth of carrots, and the abundances of the nitrochlorobenzene-degrading gene L-DEX in the soil under five groups of treatments, namely CK, V1, V2, V3, and V, were respectively: 3.54×10 5 copies·g -1 、7.81×10 7 copies·g -1 、5.22×10 8 copies·g -1 、6.48×10 8 copies·g -1 、8.53×10 8 copies·g -1 ( Figure 7 ). The abundance of the nitrochlorobenzene-degrading gene L-DEX in the soil of the V treatment group increased by three orders of magnitude compared with that of the control group (p < 0.05). The nitrochlorobenzene degradation rates of the treatment group and the control group in the soil were respectively: 1.31%, 18.36%, 9.26%, 12.79%, 98.23%( Figure 8 ).

[0050] The results showed that inoculation with phages significantly promoted the nitrochlorobenzene degradation process by soil microorganisms to a certain extent.

[0051] Example 3:

[0052] The tested potting soil was collected from the soil of a relocation site polluted by bactericide pesticides. Basic physical and chemical properties of the soil: pH 5.5, organic matter 22.4 g·kg -1 , total nitrogen 2.4 g·kg -1 , total phosphorus 0.6 g·kg -1 , total potassium 11.1 g·kg -1 , available potassium 72.7 g·kg -1 ; the mechanical composition of the soil is 52.3% sand particles (sandy soil), 20.6% silt particles, and 27.1% clay particles. The average concentration of carbon-related metabolites in the soil layer of 0 - 1 m underground is 428.2 mg·kg -1 .

[0053] A total of five groups of treatments were set up in the experiment: ① Control group (CK): 3 lettuce plants were planted in each pot (covering the seeds with 0.5 - 1 cm of soil, room temperature 25 ± 2 °C, applying 60 mg / kg of nitrochlorobenzene); ② Single phage treatment (V1): On the basis of the control group, 100 mL of a concentration of 10 6pfu·mL -1 of phage suspension; ③ single phage treatment (V2): On the basis of the control group, inoculate 100 mL of phage suspension with a concentration of 10 6 pfu·mL -1 of phage suspension; ④ single phage treatment (V3): On the basis of the control group, inoculate 100 mL of phage suspension with a concentration of 10 6 pfu·mL -1 of phage suspension; ⑤ mixed phage treatment (V): On the basis of the control group, inoculate 100 mL of the above-mentioned mixed phage suspension of the three phages with a concentration of 10 6 pfu·mL -1 . The mass ratio of the three phages in the mixture is 1:1:1. The injection bit uses a direct-push injection tube with a diameter of 2 - 5 cm, which is suitable for agricultural soil; the injection depth is 20 - 40 cm soil layer; the positioning accuracy of the robotic arm is ≤2 mm to ensure the accuracy of multi-point repeated sampling. Soil samples were taken on-site after 60 days of lettuce growth, and the abundances of the nitrochlorobenzene degradation gene L-DEX in the soil under the five treatments of CK, V1, V2, V3, and V were measured as: 3.54×10 5 copies·g -1 , 7.81×10 7 copies·g -1 , 5.22×10 8 copies·g -1 , 6.48×10 8 copies·g -1 , 8.53×10 8 copies·g -1 ( Figure 9 ). The abundance of the nitrochlorobenzene degradation gene L-DEX in the soil of the V treatment group increased by 3 orders of magnitude compared with the control group (p < 0.05). The nitrochlorobenzene degradation rates of the treatment group and the control group in the soil were measured as: 3.31%, 12.36%, 16.26%, 16.79%, 98.23%( Figure 10 ).

Claims

1. A phage composition, characterized in that, It includes three phages, all of which were deposited at the China Center for Type Culture Collection on August 1, 2018. They are Klebsiella pneumoniae phage φYSZKA, with the deposit number: CCTCC NO: M 2018513, and the taxonomic name is Klebsiella phage φYSZKA; the multivalent phage φYSZPK that attacks Klebsiella pneumoniae and Pseudomonas aeruginosa, with the deposit number: CCTCC NO: M 2018516, and the taxonomic name is Pseudomonas aeruginosa and Klebsiella phage φYSZPK; Bacillus cereus phage φYSZBA1, with the deposit number: CCTCC NO: M 2018517, and the taxonomic name is Bacillus cereus phage φYSZBA1.

2. The phage composition according to claim 1, wherein The added mass ratio of the three phages is 1:1:

1.

3. The phage composition according to claim 1, wherein The phage composition further comprises a stabilizer selected from at least one of trehalose, glycerol, gelatin or bovine serum albumin, which is used to improve the stability of the phage during storage and use.

4. The phage composition according to claim 1, wherein The phage composition comprises at least one carrier selected from at least one of sterile water, physiological saline, buffer solution or biocompatible polymer solution, which is used to facilitate the application and storage of the phage.

5. Use of the phage composition according to any one of claims 1-4 in enhancing the degradation of nitrochlorobenzene in soil.

6. The application according to claim 5, characterized in that, The soil is soil for growing wheat, lettuce or carrots.

7. The application according to claim 5, wherein The ratio of the bacteriophage composition to the soil is 10 7 - 10 8 PFU / g of phage composition.

8. Enhanced soil nitrochlorobenzene degradation improver, characterized in that, The active ingredient of the modifier is the phage composition according to any one of claims 1-4.

9. The enhanced soil nitrochlorobenzene degradation improver according to claim 8, characterized in that, It also includes auxiliary components selected from at least one of organic fertilizers, microbial inoculants, plant growth regulators or soil conditioners, which are used to synergistically enhance soil fertility and the nitrochlorobenzene degradation effect.

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