A controlled trial of BCG immunotherapy in bronchogenic carcinoma treated by surgical resection

Summary Ninety-two patients with bronchogenic carcinoma who were treated by surgical resection of the tumour were subsequently given immunotherapy with BCG (Glaxo). The study was strictly randomised into three groups. Twenty-nine patients received multipuncture BCG (50–250×10⁶ viable units) and 26 patients intradermal BCG (0.4–0.9×10⁶ viable units) treatment being given at 1, 2, 5, 9, 13 and 26 weeks after operation and every 26 weeks thereafter. Thirty-seven control patients did not receive BCG. The patients have been observed for 15–33 months. There was no significant difference in survival between the control group and the two immunotherapy groups or between the two immunotherapy groups. The tumour cell type and presence of mediastinal nodes significantly influenced overall survival but not the response to BCG immunotherapy. The possible reasons for the failure of BCG to prolong survival in this study are discussed.     

Immunotherapy of breast cancer,malignant melanoma,and acute leukemia with BCG: Prolongation of disease free interval and survival

Summary Results of immunotherapy with BCG in patients with malignant melanoma, breast cancer, and acute leukemia are described. The first study demonstrated that high doses of living BCG organisms (6×10⁸ viable units) delivered by scarification in the upper arms and legs prolonged the disease-free interval and survival of 52 malignant melanoma patients with regional lymph node metastases compared to 218 comparable surgical control patients. Patients with trunk and extremity, but not head and neck melanoma, benefited from BCG, suggesting the importance of the delivery of BCG into the tumor-involved lymphatics.The second study evaluated the therapeutic efficacy of living BCG organisms by scarification in a group of adult acute leukemia patients after the cessation of chemotherapy. Thirty-seven patients had been in remission on intermittent chemotherapy for 12–24 months. Following late intensive consolidation chemotherapy, 7 consecutive patients received no further therapy and then 30 consecutive patients received BCG. Patients maintained on BCG have had a prolonged disease-free interval compared to those given on no further therapy (P=0.07) or compared to a group of similar patients maintained on chemotherapy alone (P=0.001). Similarly, the survival has been improved for patients maintained on BCG compared to those left unmaintained (P=0.009), or those maintained on chemotherapy (P=0.001).The principles of intermittent chemotherapy combined with BCG immunotherapy, first developed in patients with disseminated melanoma and acute myelogenous leukemia, have been confirmed in a series of patients with disseminated breast cancer. Forty-five patients treated with a combination of 5-FU, adriamycin, and cyclophosphamide (FAC) plus BCG by scarification showed prolongation of remission as well as survival compared to a comparable group of 44 patients treated with FAC chemotherapy without immunotherapy. Thus, 23/44 patients treated with FAC have died (median=14 months) compared to only 5/45 patients on FAC-BCG (median=12⁺ months), P=0.005. The limitations of BCG immunotherapy as well as speculations for future developments of immunotherapy are discussed.This work was supported by Contract No1-CB 33888 from the National Institutes of Health, Public Health Service, Bethesda, Maryland 20014. Drs. Gutterman and Mavligit are the recipients of Career Development Awards (Ca 71007-02 and CA 00130-01, respectively) from the National Institutes of Health, Education, and Welfare, Bethesda, Maryland 20014.     

Binding parameters of monoclonal antibodies reacting with ovarian carcinoma ascites cells

Summary Binding parameters were determined for four mouse monoclonal antibodies reacting with three antigens on the surface of fresh human ovarian carcinoma ascites cells, under nearly physiological conditions. The object of these experiments was to aid in the selection of the optimal monoclonal antibodies for intraperitoneal immunotherapy. The number of antigenic sites per cell, the effective equilibrium association constant (affinity) and the half-life for dissociation were: for Ab MH99, 1.2×10⁶ sites/cell, (1.9–4.1)×10⁸M^–1, and 4 h; for Ab MX35, (3.2–4.1)×10⁵ sites/cell, (3.4–4.8)×10⁸M^–1, and >10 h; and for Ab MW207, 1.3×10⁵ sites/cell, (3.6–4.1)×10⁹M^–1, and 3.1 h, respectively. One of the antigens, MH99, is recognized by five different monoclonal antibodies, and competitive inhibition experiments demonstrated that two distinct determinants are present; this antigen is also recognized by the previously described Ab 17-1A. These binding data will aid the rational design of immunotherapy strategies.     

Lysis by interleukin 2-stimulated tumor-infiltrating lymphocytes of autologous and allogeneic tumor target cells

Summary Stepwise counterflow centrifugal elutriation of leukapheresed human mononuclear cells (MNC) in a Beckman JE-6B rotor and J6-M/E centrifuge yielded a population highly enriched in natural killer (NK) cells (70–75% large granular lymphocytes with 10–13 times greater NK activity) at a flow rate of 38–44 ml/min using a fixed rotor speed of 3000 rpm at 27° C. However, the mean cell recovery was <1%. To obtain sufficient numbers of purified NK cells for adoptive immunotherapy, a strategy combining counterflow centrifugal elutriation with adherence of recombinant interleukin-2(rIL-2)-activated NK cells to plastic was developed. First, MNC were elutriated to give a twofold enrichment in NK cells, containing 22% Leu19⁺ cells, 18% large granular lymphocytes and 51 lytic units of activity against K562 targets as opposed to the unfractionated MNC containing 10% Leu19⁺ cells, 7% large granular lymphocytes and 26 lytic units of activity. The mean recovery was 80±15% (n=10). Further enrichment was obtained by isolation of the elutriated cells that adhered to plastic after culture for 24 h in the presence of 1000 U/ml rIL-2. The initial adherent lymphokine-activated killer (A-LAK) cells represented 1–4% of total MNC, but their subsequent expansion was at least 10–22-fold during 8–14 days in culture with 1000 U/ml rIL-2. Using this strategy, 2 × 10⁹ normal MNC, obtained by leukapheresis, yielded 5 × 10⁸ A-LAK cells with a total of 5.7 × 10⁵ lytic units of cytotoxicity against K562 and a total of 3.3 × 10⁵ lytic units against Daudi targets. This enrichment method has yielded sufficient numbers of A-LAK cells to form the basis for a phase I clinical trial of adoptive immunotherapy in patients with advanced cancer.     

Passive transfer of tumor immunity with spleen cells from guinea pigs cured of hepatocarcinoma by nonspecific immunotherapy

Summary The purpose of this study was to evaluate cell-mediated tumor immunity in strain-2 guinea pigs cured of line-10 hepatocarcinoma by oil-in-water emulsions containing phenol-water extracts from either BCG or the Re mutant of Salmonella typhimurium (Re ET) admixed with mycobacteria glycolipid (P3). Treatment with these emulsions produced complete regression of established tumor nodules and prevented the growth of lymph node metastases in 25 of the 28 animals inoculated intradermally (ID) with 10⁶ line-10 cells and given intralesional immunotherapy 6 days later. No tumor regression was observed in animals given phenol-water extracts alone. Spleen cells, taken from guinea pigs cured of line-10 by BCG extract + P3 or Re ET + P3, were tested for their influence on tumor growth by means of an in vivo adoptive neutralization test (Winn test). Cell transfer was accomplished by the subcutanous injection of various concentrations of spleen cells admixed with 10⁵ viable line-10 cells. The results showed that as few as 10⁷ immune spleen cells completely inhibited the growth of 10⁵ tumor cells in 46–54% of the animals. The best tumor growth inhibition (77–85%) was observed in animals given 5 × 10⁷ immune cells admixed with 10⁵ tumor cells. The onset of transferrable tumor immunity was earlier in animals treated with the BCG extract + P3 than in those given the Re ET + P3. However, the duration of detectable tumor immunity was longer in the latter group. In contrast, no inhibition of tumor growth was observed in animals given spleen cells from normal or tumor-bearing guinea pigs. Moreover, spleen cells obtained from guinea pigs immunized with BCG extract + P3 or Re ET + P3 emulsions only and admixed with line-10 cells failed to transfer tumor immunity to normal animals. Thus, results from this study clearly demonstrated that cell-mediated tumor immunity was elicited in animals cured of line-10 tumor with combinations of P3 and phenol-water extracts of either BCG or Re mutant of S. typhimurium and that sensitized spleen cells effectively transferred systemic tumor immunity to normal recipients.     

A phase III comparison of methyl-CCNU + vincristine with or without BCG + allogeneic tumor cells in metastatic melanoma

Summary Sixty-two patients with metastatic malignant melanoma were randomized to treatment with either (a) methyl-CCNU (200 mg/m², PO every 8 weeks) plus vincristine (2 mg IV every 4 weeks), or (b) the same chemotherapy plus intradermal (ID) injections of irradiated (15,000 rads) allogeneic (fresh-frozen) melanoma cells (1–2×10⁸) admixed with BCG (Glaxo, 2–4.5×10⁶ organisms) every 2 weeks. Treatment cycles were repeated every 8 weeks until tumor progression. Seven (2 CR, 5 PR) objective remissions were noted among 31 patients (22.5%) treated with chemotherapy alone, whereas six (3 CR, 3 PR) objective remissions were noted among 31 patients (19%) treated with chemoimmunotherapy (P>0.05). The medians for remission duration (6 months) and survival (6.5 months) in the chemotherapy group did not differ significantly from the medians for remission duration (8 months) and survival (8 months) in the chemoimmunotherapy group. The patients manifested no unexpected toxicity. Hematologic toxicity was experienced by patients on both regimens; however, those receiving chemoimmunotherapy rebounded more quickly.     

Improved methods for investigating the external redox potential in hybridoma cell culture

Because of the interest in understanding and optimizing secretion of proteins from mammalian cells, reliable and more reproducible methods are needed to monitor the external redox potential of animal cells in suspension culture. An improved off-line method was established that greatly reduces the typically long response time of redox electrodes in cell culture media and improves the standardization of redox probes. In addition, the dependence of medium redox potential on dissolved oxygen concentrations and pH was investigated using cell-free medium. Off-line as well as on-line redox potential measurements were then applied to spinner or bioreactor cultures of murine hybridoma cells. Serum containing or protein-free medium were used. The time dependence of the experimentally determined external redox potential was found to be affected not only by oxygen, pH, and medium composition. but to a significant extent by the rate of generation of reductants by hybridoma cells. The observed specific rate of medium reduction by generation of reductants (mV h^–1 viable cell^–1) decreased during exponential growth while cell number increased from 2×10⁵ viable cells ml^–1 to 3.5×10⁶ viable cells ml^–1. This rate, however, was essentially constant at –7.3 mV h^–1±3.7 mV h^–1 per 10¹⁰ viable cells during growth under conditions of constant dissolved oxygen tension and constant pH. Using these observations, the quantity of reductants synthesized and secreted into the medium by viable hybridoma cells was estimated to be approximately 1.3 mole h^–1 per 10¹⁰ viable hybridoma cells. The time course of specific monoclonal antibody secretion rate did not correlate with changes in the external oxidation/reduction potential in either serum containing or protein-free medium.     

Microbial populations and sludge characteristics in thermophilic aerobic sewage sludge digestion

Summary Estimates of bacterial numbers from raw sewage sludge and sludge treated by thermophilic aerobic digestion were compared with simple indicators of sludge quality and concentrations of potential substrates. Significant differences were found between sludge types for all but one of the variables examined (frequency of dividing cells). During a stable period of digestor operation, the average number of viable obligate thermophiles present in digested sludge (1.63 × 10⁶ ml^–1) was approximately 10²-fold greater than in feed sludge (1.10 × 10⁴ ml^–1). Total numbers of bacteria were slightly greater in digested sludge (3.24 × 10¹⁰ ml^–1) than in feed sludge (2.39 × 10 ml^–10), as were viable counts of bacteria at incubation temperatures of 37°C and 55°C. Significant correlation was found between viable counts of bacteria at 37°C and 55°C for digested sludge, and 65°C and 55°C for feed sludge. The numbers of obligate thermophiles present and the total of bacteria present were related to the temperature and pH of the digested sludge and inversely related to the numbers ofEscherichia coli and coliforms present, which were not detected at temperatures greater than 50°C.     

Grid-generated turbulence in a mesocosm experiment

In two mesocosm experiments designed to study the effect of nutrients and turbulence on trophic levels below fish larvae in the ecosystem, turbulence was created by oscillating grids. Utilizing two different frequencies, two levels of turbulence (`high' and `low') were generated in a total of eight mesocosms in both experiments. In each mesocosm a two-layer density structure was created. We succeeded in generating turbulence that mimics turbulence found in natural ecosystems, with turbulent eddies on larger scales than those relevant for organism interactions. Energy dissipation rates () were calculated from the frequency spectrums of the measured turbulent velocities. In the second experiment the average energy dissipation rates in the upper layer were 1.9×10^–7 W kg^–1 and 5.5×10^–8 W kg^–1 in the `high' and `low' enclosures, respectively. Finding `background' turbulence levels between 5.3×10^–9 and 3×10 ^–8 W kg^–1, we suggest that future experiments consider turbulent measurements as a standard variable.     

Morphological and cultural comparison of microorganisms in surface soil and subsurface sediments at a pristine study site in Oklahoma

Surface-soil and subsurface microfloras at the site of a shallow aquifer in Oklahoma were examined and compared with respect to (1) total and viable cell numbers, (2) colony and cell types that grew on various plating media, (3) cell morphologies seen in flotation films stripped from sample particles, and (4) cellular ultrastructure. Appreciable numbers of microbial cells were present in the subsurface (total counts: 10⁶–10⁷ cellsg^–1; viable counts up to 10⁶ cells · g^–1), but the subsurface microflora was considerably less populous than that of the surface soil (total counts: 10⁹ cells·g^–1; viable counts: 10⁷–10⁸ cells · g^–1). The subsurface microflora (especially that of the saturated zone) also appeared to be much less diverse, containing fewer microbial types that would grow on enumeration plates (on nutrient-rich media, 3–4 colony types versus 19–22 for the surface soil) and fewer cell types that could be distinguished by direct microscopy (3–4 types versus 17 for the surface soil). The specific types of microorganisms that were numerically predominant in the aquifer sediments were entirely different from those that were predominant in the surface soil. Moreover, the predominant types varied from one depth to another within the saturated zone. The potential metabolic capability of the subsurface microflora, as indicated by its readiness to grow rapidly on nutrient-rich media, also varied with depth.     

The bacterial biota on crustose (nonarticulated) coralline algae from Tasmanian waters

The bacterial biota associated with the cuticle surface of healthy benthic samples of crustose nonarticulated coralline algae from the east coast of Tasmania (Australia) was examined by bacteriological cultivation and electron microscopy. In 32 samples studied, the viable count on Zobell's marine agar (supplemented with vitamins) was 3.3×10⁶ bacteria g^–1 wet wt. (range 2.9×10⁴–2.7×10⁷). Of 732 strains isolated from 16 out of 32 samples and identified to genus level,Moraxella was the predominant genus (66%). In contrast,Moraxella comprised only 11% of 217 strains isolated from benthic seawater samples collected at the same time as coralline algae. In 22 out of 32 algal samples examined by scanning electron microscopy, the total count was 1.6 × 10⁷ bacteria g^–1 wet wt. (range 5.1× 10⁶–3.8×107); the major morphotype was cocco-bacilli (80%). Several environmental factors did not significantly influence the viable count or generic distribution, or the total count or morphotypic distribution of bacteria on the cuticle. These factors included geographical site, season, storage of samples in aquarium conditions, and the presence or absence of abalone from shells that the coralline algae encrusted. The microbiota, consisting mostly of the nonmotile bacterial genusMoraxella, appeared to be highly adapted to its calcerous plant host.     

The endogenous metabolism ofFibrobacter succinogenes and its relationship to cellobiose transport,viability and cellulose digestion

Fibrobacter succinogenes S85 digested ballmilled cellulose at a rapid rate (0.10 h^–1), but there was a long lag time if the culture was not transferred daily. WhenF. succinogenes was starved for 100h, a large fraction of the cells (>30%) still bound to cellulose, but the lag time was 150h. The lag time was similar for either cellulose- or cellobiose-grown inocula, and lag times were highly correlated (r ² = 0.91) with a decrease in viable cell number. The number of viable cells declined from 10⁸ to 10⁶ in the first 30h of starvation, and by 72h the viable cell number was less than 10³/ml. Cells growing exponentially on cellobiose had a large pool of polysaccharide, and continuous culture experiments indicated that polysaccharide accumulation was not significantly influenced by the growth rate of the culture (approximately 0.7 mg polysaccharide mg^–1 protein). When the cellobiose was depleted, cellular polysaccharide decreased at first order rate of 0.09 h^–1. The rate of endogenous metabolism was initially 0.08mg polysaccharide mg^–1 protein h^–1, and there was little decline in viability until the rate of endogenous metabolism was less than 0.01 mg polysaccharide mg^–1 protein h^–1. When the rate was less than 0.01 mg polysaccharide mg^–1 protein h^–1, the cells could not maintain a sodium gradient, transport cellobiose or grow. The endogenous metabolic rate needed for cell survival was 20 fold less than the maintenance energy of cells growing in continuous culture (0.01 versus 0.232mg carbohydrate mg^–1 protein h^–1).     

Immune responsiveness to Ambrosia artemishfolia (short ragweed) pollen allergen Amb a VI (Ra6) is associated with HLA-DR5 in allergic humans

The relationship between HLA type and specific immune responsiveness toward ultrapure Ambrosia artemisiifolia (short ragweed) pollen allergen Amb a VI (Ra6) was explored in a genetic-epidemiologic study of groups of 116 and 81 Caucasoid subjects who were skin-test \ positive (ST^–) toward common environmental allergens. Specific immune responsiveness to Amb a VI was assessed by measuring serum IgE and IgG antibodies (Abs) by double Ab radioimmunoassay in both ST^– groups. Significant associations were found between IgE Ab responsiveness to Amb a VI and the possession of HLA-DR5; P values for the two groups were, respectively, 7 × 10^–7 and 1 × 10^–3 by nonparametric analyses, and 4 × 10^–11 and 5 × 10^–8 by parametric analyses. The levels of significance for the associations between HLA-DR5 and IgG Ab responsiveness were highly dependent on the extent of ragweed immunotherapy (Rx) within the patient group; by parametric statistics, the associations were 10^–11 for the group that had received relatively little Rx and 2 × 10^–3 for the group that had received more intensive Rx. These results provide further striking evidence for the existence of specific HLA-linked human Ir genes involved in responsiveness toward inhaled allergens and illustrate the usefulness of the allergy model in studies of the genetic basis of human immune responsiveness. Extension of these studies to investigation of structure-function relationships involved in antigen recognition by Ia molecules and the T-cell receptor will lead to a better understanding of human susceptibility toward immunologic diseases.Abbreviations used in this paper Ab antibody - Amb a VI Amb a V, new IUIS nomenclature for Ambrosia artemisiifolia pollen allergens nos. 6 and 5 (short ragweed Ra6 and Ra5) (Marsh et al. 1986b) - Lol p II, III new IUIS nomenclature for Lolium perenne pollen allergens II and III (perennial rye grass, Rye II and Rye III) (Marsh et al. 1986b) - BBS borate-buffered physiologic saline - BSA bovine serum albumin - DARIA double-antibody radioimunoassay - Ia immune-associated - PAGE polyacrylamide gel electrophoresis - RIST radioimmunosorbent test - Rx immunotherapy - SDS sodium dodecyl sulfate - ST skin test     

Colonization potential of bacteria in the rhizosphere

The effect of inoculum density on growth and steady-state populations of aPseudomonas sp., aMycoplana sp., and aCurtobacterium sp. in the rhizosphere if gnotobiotic barley plants was studied. Inoculation of sterile barley seedling at concentrations of about 1×10³, 1×10⁵ and 1×10⁷ viable cells (mg dry wt root)^–1 resulted in rapid colonization; maximum populations of about 5×10⁷ viable cells (mg dry wt root)^–1 developed in each case. We define this maximum population as the colonization potential. Measurement of growth of known rhizosphere bacteria might be a useful index of the amount of available carbon and energy lost by growing roots.     

Influence of tetracycline on the submerge Pleurotus saca cultivation

Summary The influence of various tetracycline hydrochlorides (TC HC concentrations: 3,6,8 and 12 × 10^–3g 1^–1), on growth and protection against bacterial infections in submerged Pleurotus saca cultivation was studied. 8 × 10^–g 1^–1 of TC HC added before the inoculation showed a reasonable protection effect, simultaneously promoting the growth of fungus. In the presence of tetracycline the lag phase was prolonged, and the final biomass production increased by 200 % on average. If the infection occurred during the cultivation process, even 12–16 × 10^–3g 1^–1 of added TC HC, could not prevent bacterial infection.     

Expansion and neural differentiation of embryonic stem cells in adherent and suspension cultures

The embryonic stem cell line, S25, is a genetically modified line that allows lineage selection of neural cells (M. Li, L. Lovell-Badge, A. Smith (1998) Current Biology 8: 971–974). Here, the growth parameters of this cell line were analysed. Serial passaging in adherent conditions enabled these cells to grow rapidly (average specific growth rates of 0.035 h^–1) and generate high viable cell densities (above 90%). The aggregation of the S25 cells into embryoid bodies (EBs) was also studied, indicating limited cell growth (maximum cell densities of 2.7×10⁵ cells ml^–1) and a high variability of aggregate size (70–400 m after 8 d). Enzymatic dissociation of EBs with 1% (v/v) trypsin gave highest cell viability (91%) and density (1.4×10⁴ cells ml^–1) and the cells thus obtained are able to differentiate into neurons.     

Effect of high concentration of nutrients onBacillus thuringiensis cultures

Summary Bacterial insecticide production using a strain ofBacillus thuringiensis var. kurstaki was studied in batch culture considering the influence of increasing concentration of components of a glucose — yeast extract — mineral salts medium.It was found that spore counts were increased from 1.08×10¹² spores. 1^–1 to 7.36×10¹² spores. 1^–1 and toxin level from 1.05 mg.ml^–1 to 6.85 mg.ml^–1, when the concentration of glucose was increased from 8 to 56 (g 1^–1), with the corresponding increase in the rest of medium components. Higher concentration of nutrients inhibit either spore count or toxin production.Preliminary experiments of fed-batch cultures which allows the use of high amounts of nutrients were also carried out. In this case spore counts of 1.2×10¹³ spores.1^–1 were achieved.     

Communicating junctions and calmodulin: Inhibition of electrical uncoupling inXenopus embryo by calmidazolium

Summary This paper reports the inhibitory effects of calmidazolium (CDZ), a calmodulin inhibitor, on electrical uncoupling by CO₂. Membrane potential and coupling ratio (V ₂/V₁) are measured in two neighboring cells ofXenopus embryos (16 to 64 cell stage) for periods as long as 5.5 hr. Upon exposure to 100% CO₂, control cells consistently uncouple even if the CO₂ treatments are repeated every 15 min for 2.5 hr. CDZ (5×10^–8–1×10^–7 m) strongly inhibits uncoupling. The inhibition starts after 30, 50 and 60 min of treatment with 1×10^–7, 7×10^–8 and 5×10^–8 m CDZ, respectively, is concentration-dependent and partially reversible. In the absence of CO₂, CDZ also improves electrical coupling. CDZ has no significant effect on membrane potential and nonjunctional membrane resistance. These data suggest that calmodulin or a calmodulin-like protein participates in the uncoupling mechanism.     

Canine osteosarcoma: Results of amputation with and without adjuvant immunotherapy

Summary Forty-eight dogs with spontaneously occurring osteogenic sarcoma (OS) were treated by amputation. Six dogs died soon after surgery, 34 dogs died or were euthanized 1–11 (median 4) months after, and eight dogs (17%) are alive 11–47 (median 21) months after amputation. Thirty-one dogs did not receive any therapy after amputation. Seventeen dogs received postamputation adjuvant immunotherapy consisting of intradermal bacillus Calmette-Guerin and an autologous tumor homogenate preparation administered every other week for 12 weeks. Twelve of these dogs were included in a prospectively randomized therapeutic trial in which a control group of 11 dogs received no therapy after amputation. Neither disease-free interval nor survival was prolonged by the immunotherapy protocol. Nevertheless, the current study demonstrates the feasibility of studying naturally occurring canine OS as a clinical and therapeutic model for OS in man.     

Effect of microbial concentration on biodegradation rates of phenols

Summary Biodegradation rates of 12 phenols were measured with respect to acclimated microbial biomass ranging from 2.3×10⁴ to 2.3×10⁸ cells/l. Rates ranged between 0.02 mg l^–1 day^–1 for 1.6 mg/lp-bromophenol exposed to 2.3×10⁴ cells/l and 1.41 mg l^–1 day^–1 for 3.2 mg/lp-methylphenol exposed to 2.3×10⁸ cells/l. Generally, rates for all phenols were first-order in substrate concentration and zero-order in biomass concentration. Bromophenol biodegradation was preceded by lag periods of varying lengths and to a small extent the rate was dependent on microbial biomass. Results from this study suggest chemical biodegradation generally exhibits pseudo-first-and occasionally, second-order kinetics.