Lu Guanghua, Tang Jie ^#, Yuan Xing, Zhao Yuanhui
(Department of Environmental Science, Northeast Normal University, ChangChun 130024; ^#Department of Environmental Science and Technology, Jilin University)

Received Feb.16, 2001; Supported by the National Natural Science Foundation of China (29877004)

Abstract The biodegradability of 47 substituted benzenes was determined by BOD technique. The molecular weight (M_w), the total surface area (TSA), the energy of the highest occupied molecular orbital (E_HOMO), the heat of formation(H_f), and the moment of dipole (m) of 47 studied compounds were calculated by the quantum chemical method MOPAC6.0-AM1. The ionization constant (pK_a) and n-octanol/water partition coefficient (logP) were obtained from Qsar software and Biobyte software, respectively. The quantitative structure-biodegradability relationship studies were developed by linear regression analysis. The correct prediction rate of obtained model is up to 85% for the testing set. It has been shown that the biodegradability of substituted benzenes in natural river water is mainly related to electronic parameter E_HOMO, H_f and steric parameter M_w.
Keywords structure parameter, BOD/ThOD, correlation, QSBR model,  prediction

1. INTRODUCTION   
Most substituted benzenes constitute a class of important environmental pollutants that are present in the Songhua River of Jilin Province, China. The presence of many of these chemicals in the natural waters is a serious public health problem. Biodegradation is an important mechanism for removing them from ecosystem. Biodegradation can eliminate hazardous chemicals, we can presume, Via a series of enzymatic processes in microorganisms, into innocuous forms, degrading them by mineralization to carbon dioxide and water^[1].Biochemical oxygen demand (BOD) is the relatively simple method of determination and the information obtained is very useful for most cases. So the parameter is used to determine the ultimate degradability, which is demanded in most of the standard tests^[2]. However, gathering this information is labor intensive, time consuming, and expensive due to the large number of these chemicals. Therefore, it is necessary to develop correlation and predictive techniques in order to estimate biodegradability. The quantitative structure-biodegradability relationships (QSBRs) have been used to predict the fate of organic chemicals and analyse biodegradation mechanism^[3].
    At present, the reports on QSBR studies of organic chemicals are not so much for it is not easy to gain biodegradation data at the same experimental conditions. The biodegradability of 47 organic pollutants was determined by taking the bacteria in the Songhua River as inoculum, and QSBR models were developed using the structure parameters of chemicals as descriptors in this paper.

2. MATERIALS AND METHODS    
Organic and inorganic chemicals were of analytical and reagent grade, respectively. Formula of inorganic salt stock medium (mg/l): K₂HPO₄ 21.75, KH₂PO₄ 8.5, Na₂HPO₄·7H₂O 33.4, NH₄Cl 1.7, CaCl₂ 27.5, FeCl₃·6H₂O 0.25, MgSO₄·7H₂O 22.5. Water sample was gathered from Jilin section in the Songhua River. Temperature of the water sample: 15-20℃; pH: 6.8-7.0; Dissolve oxygen (DO): about 8.0 mg/l. The bacteria counts were determined by standard plate count techniques^[4] , and are about 800-3000/ml. Biodegradation of studied compounds was determined using the BOD technique^[5]. The initial test concentration of chemicals was planned at 2mg/l on the basis of their acute toxicity to aquatic organisms, theoretical oxygen demand (ThOD), and residual DO of at least 1mg/l at the final day.
    The 1ml above inorganic salt stock solution was added to 1L river water sample as soon as the River water was gathered. The test chemical was added to 250ml BOD bottles, then the bottles were filled to capacity with the water sample, sealed, and incubated for 5 days at 20°1±C.There were two replicates at each test compound and each control (only inoculum). The DO concentrations were determined by the iodometric titration method ^[5].
    The biodegradability was expressed as BOD% by comparing the measured BOD₅ with ThOD, which is calculated from the molecular formula of the test compound (Table 1).

Table1. The experimental and predicted BOD % of 47 substituted benzenes

Biode.¹and Biode.²are the qualitative estimation for experimental and predicted BOD%, respectively; andImeans non-readily biodegradable, II biodegradable, and III readily biodegradable.

2.CALCULATION METHODS   
The molecular weight (M_w), the total surface area (TSA), the energy of the highest occupied molecular orbital (E_HOMO), the heat of formation(H_f), and the moment of dipole (m) of 47 substituted benzenes were calculated by the quantum chemical method MOPAC6.0-AM1 on energy-minimized structures. The ionization constant (pK_a) and n-octanol/water partition coefficient (logP) were obtained from Qsar software and Biobyte ClogP for Window software, respectively. The parameter values of 47 compounds were listed in Table 2. All statistical analyses were carried out using SPSS package.

Table2.The structure parameters of 47 substituted benzenes

*The compound number in Table 2 is as same as in Table 1.

3.RESULTS AND DISCUSSION
Biodegradation of a chemical in the aquatic environment is predominantly through microbial attack, and thus, we may presume, via enzymatic processes^[6]. In general, the factors determining the rate of biodegradation can be divided into two kinds. (1) The uptake rates and transport rates (e.g., the uptake rates by microbial cells or transport rates within the cell to the relevant enzymes). (2) The rates binding to the active site of an enzyme, and/or by the rate at which they undergo enzymatic transformation. In the absence of a specific uptake mechanism, organic compounds are probably transported into bacterial cells by passive diffusion through the lipid membrane. If the diffusion of chemicals in cell membrane and water belongs to partition process, the diffusion coefficient should be in directly proportional to logP. Therefore, biodegradation rates should be related to macroscopic hydrophobic parameters if diffusion and uptake are rate-limiting step of biodegradation. The enzyme-catalyzed transformation of a compound occurs by its binding to the site of the enzyme through the formation of hydrogen or covalent bonds. The strength of this interaction is influenced by the electronic structure and the steric structure of compound coinciding with the active site of enzyme. So, if binding to enzyme or transformation is rate-limiting step, biodegradation rate of compounds should be related to the factors influencing the binding or reacting with enzyme (e.g. steric and/or electronic parameters).
    Pitter et al^[7]respectively developed QSBR models for the biodegradation rate V of ortho-, meta-, and para- substituted phenols using the substituent electron parameter s: logV=-0.430s+1.7，n=5(ortho-)，R=0.980，s=0.040 (1) ; logV=-0.616s+1.72，n=4 ( meta-)，R=0.960，s=0.102 (2) ; logV=-0.323s+1.65，n=5 ( para-)，R=0.990，s=0.083 (3). Pitter's results showed that the biodegradation rate V of substituted phenols is influenced mainly by the electron effect of substituent. Yet there is not linear correlation between V and steric parameters or hydrophobic parameters.
    Damborsky and Schultz^[8] developed and compared QSBR models for biodegradability of groups of m-anilines and p-phenols. Anilines were degraded by an unknown bacterial strain isolated from the Onconee River and phenols by Pseudomonas putida U. Biodegradability was expressed as the second-order kinetics rate constant (K_b). The QSBR equations were obtained as follows:
logK_b=-11.237r_w+0.0092M_w+0.374pK_a-14.194, n=7(anilines), R²=0.953 (4); logK_b=-13.743r_w+0.0351V_w+0.195pK_a-13.462, n=8(phenols), R²=0.986 (5);
logK_b=-11.233r_w +0.315pK_a-12.738, n=15(anilines and phenols), R²=0.986 (6).
    In which, r_w is Van der Waal's radii; V_w is Van der Waal's volume; M_w is molecular weight; and pK_a is ionization constant. Damborsky and Schultz thought that electronic and steric or size properties should be necessary for modeling biodegradation.
    Boethling^[9] developed correlation equations for biodegradability, the second order kinetics rate constant (K_b) of 12 Phthalic acids and phthalate esters respectively using M_w and logP: K=-0.977×10^-3 M_w +0.533，n=12，R=-0.954 （7）；K=-0.0243logP+0.395，n=12，R=-0.931（8）. The results showed that the biodegradability of studied compounds appears to be negative correlation with molecular size and hydrophobicity.
    In addition, Lu et al^[10] also developed quantitative structure-biodegradability relationship model on the maximum specific removal rates (Q_TOD) with the structure parameters as follows: Q_TOD= -0.614M_W -9.704E_HOMO-0.129 H_f , n=52, R²=0.860, SE=14.89, F=100.21, P=0.000（9）. Lu concluded that the  biodegradation of studied compounds is related mainly to steric parameter and electronic parameter.
    Based on the above work, M_w, TSA, E_HOMO, H_f, m, pK_a and logP are selected as the structure parameters to establish the QSBR models. 47 compounds are randomly divided into two sets. Of which, 40 compounds are included in the training set and 7 compounds in the testing set. By the stepwise regression analyses, two QSBR models are developed by the degradation data (BOD%) and parameters of studied chemicals and listed in Table 3.

Table 3. The results of stepwise regression analyses

As the constant item is contained in Model 1, its S.E. is very high, t value very small and Sig. (0.562) is much higher than 0.05. So the constant item is removed from QSBR models just as Model 2. Model 1-2 can be expressed as equation (10) - (11) respectively as follows:
BOD%=18.50-13.858E_HOMO-0.465H_f-0.924MW, n=40, R²=0.735, S.E.=13.43, F=33.25, Sig.=0.000 (10)
BOD%=-16.091E_HOMO-0.452H_f-0.937MW, n=40, R²=0.939, S.E.=13.31, F=188.69, Sig.=0.000       (11)
    There, S.E. is the standard error; t is the t-test value; Sig. is the significant level; R² is the square of correlation coefficient; F is the mean square ratio; and n is the number of compounds. Obviously, the correlation is more obvious and the standard error is lower in equation (11). So this equation is used to predict the biodegradability (Table 1).
    The biodegradability of 47 substituted benzenes may be divided into three kinds according to their BOD% values:I.non-readily biodegradable (BOD% £ 30), II.biodegradable (30<BOD%<60), and III.readily biodegradable (BOD% ³ 60). If the rule is used to qualitatively estimate the predicted results, the correct prediction rate of model 2 is near to 80% for the training set, while is more than 85% for the testing set. This result shows the obtained QSBR model fits well, especially for the testing set.
    The obtained QSBR models show that the biodegradation of studied compounds is related mainly to electronic parameter E_HOMO and H_f, and steric parameter M_w. E_HOMO is the energy of the highest occupied molecular orbital, and it is related to ionization potential^[11]. The higher the -E_HOMO values, the higher the BOD% values, the easier the compound is degraded by river water bacteria. The heat of formation (H_f) of a compound is a measure of its stability, and hence would be expected to reflect the ability of the compound to biodegrade^[3]. The lower the H_f value, the easier the chemical is degraded by microbes. M_w is the molecular weight, and it can reflect the size of a molecular. The smaller the molecular, the smaller the steric resistance in microorganisms, the easier the molecular penetrate into the cell through its cell membrane and arrive at the active site of an enzyme. Therefore, the smaller the M_w, the easier the compound is biodegraded. This result is consistent with Boethling's^[9] and Lu's^[10].

4.CONCLUSION   
The biodegradability of 47 substituted benzenes was determined by BOD technique; QSBR studies were developed for BOD% using -E_HOMO, H_f and M_w as structure parameters by linear regression analysis method. The correct prediction rate of obtained model is up to 85% for the testing set and the total correct prediction rate is up to 80%. It has been shown that the biodegradability of studied compounds is related mainly to electron transfer, stability and size of molecular.

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[4] Wang Jialing. Environmental Microbiology, Beijing: Higher Education Press, 1988, 12.
[5] American Public Health Association. Standard Methods for Examination of Water and Wastewater. 15ed. Washington, D.C.: American Public Health Association,  1980, 70.
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