A study of the effects of fungitoxic compounds on Phytophthora cinnamomi in water

Copper and chlorine-releasing compounds were the most fungitoxic of 13 compounds tested in water for inhibition of Phytophthora cinnamomi. Mycelium was killed when immersed for 24 h in suspensions containing copper (13–45 mg/1) or a solution containing free residual chlorine (100 mg/1). Sub-lethal concentrations of these compounds reduced the numbers of sporangia. Exposing zoospores of P. cinnamomi for 60 s to water containing 2 mg free residual chlorine/1 reduced subsequent colony production on agar plates by 96–100%.
Prothiocarb, etridiazole ex. and furalaxyl killed mycelium immersed in solutions or suspensions for 3–6 days at 1500, 1000 and 600 mg a.i./l respectively and suppressed sporangium production at 1000, 500 and 300 mg/1.
Mycelium survived 3 days' immersion in ethyl hydrogen phosphonate compounds at 4000 mg a.i./l but 1000 mg a.i./l suppressed sporangium formation.
1-(2-Cyano-2-methoxyiminoacetyl)-3-ethyl urea and drazoxolon did not kill mycelium at 2000 and 1500 mg a.i./l respectively with a 6-day exposure, but reduced numbers of sporangia produced.     

Total D-amino and other free amino acids increase in the muscle of crayfish during seawater acclimation

Freshwater crayfish contained smaller amounts of total free amino acids in the muscle than marine prawns and lobsters. In the muscle of seawater acclimated crayfish, however, total amino acids were elevated by 3.7-fold and reached almost equivalent values to those commonly found in marine species. Glycine, alanine, proline, asparagine, and glutamine were the major amino acids elevated during seawater acclimation. Changes in alanine were most pronounced with its concentration increasing by 112 mmol/kg water (15.2-fold approx.). Crayfish muscle contains significant amounts of D-amino acids (mostly D-alanine) and these rose from 1.85 to 58.5mmol/kg water during seawater acclimation. D-Alanine in the seawater crayfish corresponds to almost one half of the total (D + L) alanine pool.     

Bactericidal properties of an organic N-chloramine formed in situ

Agent I (3-chloro-4,4-dimethyl-2-oxazolidinone) formed in situ was compared with pre-formed agent I as a disinfectant against Staphylococcus aureus. In situ formation involved combining the non-chlorinated oxazolidinone precursor with calcium hypochlorite to form 5 and 10 mg/l total chlorine concentrations of agent I. The variables included in the study were temperature, pH and concentration. Overall the bacteria were killed more rapidly at 22° than at 4°C. The in situ formation appeared to occur most rapidly at pH 7˙0, slightly slower at pH 9˙5, and very slowly at pH 4˙5 as evidenced by the presence of residual free chlorine. In the in situ experimental runs the 5 and 10 mg/l concentrations were equally effective in obtaining a six log decline in cfu/ml. This study indicates the potential for using the organic N-chloramine as a general purpose disinfectant while omitting the laboratory synthesis of the final product.     

Re‐examining extreme longevity of the cave crayfish Orconectes australis using new mark–recapture data: a lesson on the limitations of iterative size‐at‐age models

1. Centenarian species, defined as those taxa with life spans that frequently exceed 100 years, have long been of interest to ecologists because they represent an extreme end point in a continuum of life history strategies. One frequently reported example of a freshwater centenarian is the obligate cave crayfish Orconectes australis, with a maximum longevity reported to exceed 176 years. As a consequence of its reported longevity, O. australis has been used as a textbook example of life history adaptation to the organic‐carbon limitation that characterises many cave‐stream food webs. 2. Despite being widely reported, uncertainties surround the original estimates of longevity for O. australis, which were based on a single study dating from the mid‐1970s. In the present study, we re‐evaluated the growth rate, time‐to‐maturity, female age‐at‐first‐reproduction and longevity of O. australis using a mark–recapture study of more than 5 years based upon more than 3800 free‐ranging individuals from three isolated cave streams in the south‐eastern United States. 3. The results of our study indicate that accurate estimates of the longevity of O. australis are ≤22 years, with only a small proportion of individuals (<5%) exceeding this age. Our estimates for female time‐to‐maturity (4–5 years) and age‐at‐first‐reproduction (5–6 years) are also substantially lower than earlier estimates. 4. These new data indicate that the age thresholds for life history events of O. australis are comparable to other estimates for a modest assemblage of cave and surface species of crayfish for which credible age estimate exists, suggesting that a cave environment per se is not required for the evolution of extreme longevity in crayfish.     

Determinants of the microbiological characteristics of South Australian swimming pools.

A survey of 100 swimming pools has been conducted to assess the effectiveness of disinfection practices against various microorganisms and to check compliance with recommended chlorine levels and pH. Microbiological quality was assessed from densities of total coliforms, Escherichia coli, and Pseudomonas aeruginosa, total colony counts, and the presence or absence of amoebae, including the pathogen Naegleria fowleri. Although a free chlorine residual of 1.0 mg/liter and a pH range of 7.0 to 7.6 are recommended by local health authorities, 41 pools had a lower free chlorine residual and 37 had a pH outside this range. Multiple logistic regression analysis was used to test the association of field measurements with the microbiological data. The analysis demonstrated a strong positive association of free chlorine with bacteriological quality and the absence of Naegleria spp. No other field measurement was predictive in this regard, although the absence of all amoebae was associated with a relatively low water temperature and pH. Using a mathematical model derived from this analysis, it was estimated that 99% of pools would have acceptable bacteriological quality and 94% would be free of Naegleria spp. at a free chlorine residual of 1.0 mg/liter. However, at the mean water temperature (23 degrees C) and pH (7.5) seen in this study, other amoebae would still be detectable in 500-ml samples taken from 40% of pools at this chlorine level.     

Effects of Assimilable Organic Carbon and Free Chlorine on Bacterial Growth in Drinking Water

Assimilable organic carbon (AOC) is one of the most important factors affecting the re-growth of microorganisms in drinking water. High AOC concentrations result in biological instability, but disinfection kills microbes to ensure the safety of drinking water. Free chlorine is an important oxidizing agent used during the disinfection process. Therefore, we explored the combined effects of AOC and free chlorine on bacterial growth in drinking water using flow cytometry (FCM). The initial AOC concentration was 168 μg.L^-1 in all water samples. Without free chlorine, the concentrations of intact bacteria increased but the level of AOC decreased. The addition of sodium hypochlorite caused an increase and fluctuation in AOC due to the oxidation of organic carbon. The concentrations of intact bacteria decreased from 1.1×10⁵ cells.mL^-1 to 2.6×10⁴ cells.mL^-1 at an initial free chlorine dose of 0.6 mg.L^-1 to 4.8×10⁴ cells.mL^-1 at an initial free chlorine dose of 0.3 mg.L^-1 due to free chlorine originating from sodium hypochlorite. Additionally, free chlorine might be more obviously affected AOC concentrations than microbial growth did. These results suggested that AOC and free chlorine might have combined effects on microbial growth. In this study, our results showed concentrations determined by FCM were higher than those by HPC, which indicated that some E. coli detected by FCM might not be detected using HPC in drinking water. The level of free chlorine might restrain the consumption of AOC by inhibiting the growth of E. coli; on the other hand, chlorination might increase the level of AOC, thereby increase the potential for microbial growth in the drinking water network.     

Determinants of the microbiological characteristics of South Australian swimming pools

A survey of 100 swimming pools has been conducted to assess the effectiveness of disinfection practices against various microorganisms and to check compliance with recommended chlorine levels and pH. Microbiological quality was assessed from densities of total coliforms, Escherichia coli, and Pseudomonas aeruginosa, total colony counts, and the presence or absence of amoebae, including the pathogen Naegleria fowleri. Although a free chlorine residual of 1.0 mg/liter and a pH range of 7.0 to 7.6 are recommended by local health authorities, 41 pools had a lower free chlorine residual and 37 had a pH outside this range. Multiple logistic regression analysis was used to test the association of field measurements with the microbiological data. The analysis demonstrated a strong positive association of free chlorine with bacteriological quality and the absence of Naegleria spp. No other field measurement was predictive in this regard, although the absence of all amoebae was associated with a relatively low water temperature and pH. Using a mathematical model derived from this analysis, it was estimated that 99% of pools would have acceptable bacteriological quality and 94% would be free of Naegleria spp. at a free chlorine residual of 1.0 mg/liter. However, at the mean water temperature (23 degrees C) and pH (7.5) seen in this study, other amoebae would still be detectable in 500-ml samples taken from 40% of pools at this chlorine level.     

Assessment of bismuth thiols and conventional disinfectants on drinking water biofilms

AIMS: Biofilms in water distribution systems represent a far more significant reservoir of micro-organisms than the water phase. Biofilms are (i) resistant to disinfectants, (ii) nuclei for microbial regrowth, (iii) a refuge for pathogens, (iv) accompanied by taste and odour problems, and (v) corrode surfaces. The effects of the current strategies for disinfection of drinking water systems in large buildings (chlorination, copper and silver ionization, and hyper-heating) were compared with a new generation of bismuth thiol (BT) biocides. METHODS AND RESULTS: Multispecies biofilms were treated with 0.8 mg l(-1) of free chlorine, 400 and 40 microg l(-1) of copper and silver ions, respectively, at 55 and 70 degrees C, and bismuth-2,3-dimercaptopropanol (BisBAL). Furthermore, the effect of combined heat and BisBAL on planktonic cell viability was examined in monoculture using Escherichia coli suspensions. Inactivation rates for BisBAL were similar to copper-silver ions, where the effects were slower than for free chlorine or temperature. The BisBAL effect on E. coli monocultures was augmented greatly by increasing temperatures. CONCLUSIONS: Like copper-silver ions, BTs show more persistent residual effects than chlorine and hyper-heating in water systems. BT efficiency increased with temperature. Like copper-silver ions, BT action is relatively slow. SIGNIFICANCE AND IMPACT OF THE STUDY: BT presents a new approach to containing water biofilms. BT action is not as rapid, but is more thorough than chlorine, and less caustic. BTs may also be more efficacious in hot water systems. At sub-minimum inhibition concentration levels, BTs uniquely inhibit bacterial exopolysaccharide, thereby retarding biofilm formation. Thus, the combination of bactericidal and residual effects may prevent slime build-up in hot water systems.     

Bactericidal properties of an organic N-chloramine formed in situ

Agent I (3-chloro-4,4-dimethyl-2-oxazolidinone) formed in situ was compared with pre-formed agent I as a disinfectant against Staphylococcus aureus. In situ formation involved combining the non-chlorinated oxazolidinone precursor with calcium hypochlorite to form 5 and 10 mg/l total chlorine concentrations of agent I. The variables included in the study were temperature, pH and concentration. Overall the bacteria were killed more rapidly at 22 degrees than at 4 degrees C. The in situ formation appeared to occur most rapidly at pH 7.0, slightly slower at pH 9.5, and very slowly at pH 4.5 as evidenced by the presence of residual free chlorine. In the in situ experimental runs the 5 and 10 mg/l concentrations were equally effective in obtaining a six log decline in cfu/ml. This study indicates the potential for using the organic N-chloramine as a general purpose disinfectant while omitting the laboratory synthesis of the final product.     

A note on legionellas in whirlpools

Water samples from 52 whirlpools (jacuzzi), water temperature 35–40°C, and from 50 swimming pools, water temperature 8–30°C, were investigated for the presence of Legionella pneumophila. This was isolated from 11 of 28 whirlpools with free available chlorine less than 0.3 mg/1. No legionellas were detected in 23 whirlpools with free available chlorine over 0.3 mg/l. Legionella pneumophila was found in two swimming pools. The results indicate that 0.3 mg/l of free available chlorine is sufficient to eliminate legionellas from whirlpools.     

Chlorine resistance patterns of bacteria from two drinking water distribution systems.

The relative chlorine sensitivities of bacteria isolated from chlorinated and unchlorinated drinking water distribution systems were compared by two independent methods. One method measured the toxic effect of free chlorine on bacteria, whereas the other measured the effect of combined chlorine. Bacteria from the chlorinated system were more resistant to both the combined and free forms of chlorine than those from the unchlorinated system, suggesting that there may be selection for more chlorine-tolerant microorganisms in chlorinated waters. Bacteria retained on the surfaces of 2.0-microns Nuclepore membrane filters were significantly more resistant to free chlorine compared to the total microbial population recovered on 0.2-micron membrane filters, presumably because aggregated cells or bacteria attached to suspended particulate matter exhibit more resistance than unassociated microorganisms. In accordance with this hypothesis, scanning electron microscopy of suspended particulate matter from the water samples revealed the presence of attached bacteria. The most resistant microorganisms were able to survive a 2-min exposure to 10 mg of free chlorine per liter. These included gram-positive spore-forming bacilli, actinomycetes, and some micrococci. The most sensitive bacteria were readily killed by chlorine concentrations of 1.0 mg liter-1 or less, and included most gram-positive micrococci, Corynebacterium/Arthrobacter, Klebsiella, Pseudomonas/Alcaligenes, Flavobacterium/Moraxella, and Acinetobacter.     

Chlorine decay and bacterial inactivation kinetics in drinking water in the tropics

The decay of free chlorine (Cl₂) and combined chlorine (mostly monochloramine: NH₂Cl) and the inactivation of bacteria was examined in Dar es Salaam, Tanzania. Batch experiments, pilot-scale pipe experiments and full-scale pipe experiments were carried out to establish the kinetics for both decay and inactivation, and to compare the two disinfectants for use under tropical conditions. The decay of both disinfectants closely followed first order kinetics, with respect to the concentration of both disinfectant and disinfectant-consuming substances. Bacterial densities exhibited a kinetic pattern consisting of first order inactivation with respect to the density of the bacteria and the concentration of the disinfectant, and first order growth with respect to the bacterial density. The disinfection kinetic model takes the decaying concentration of the disinfectant into account. The decay rate constant for free chlorine was 114 lg^-1h^-1, while the decay rate constant for combined chlorine was 1.84 lg^-1h^-1 (1.6% of the decay rate for free chlorine). The average concentration of disinfectant consuming substances in the water phase was 2.6 mg Cl₂/l for free chlorine and 5.6 mg NH₂Cl/l for combined chlorine. The decay rate constant and the concentration of disinfectant consuming substances when water was pumped through pipes, depended on whether or not chlorination was continuous. Combined chlorine especially could clean the pipes of disinfectant consuming substances. The inactivation rate constant , was estimated at 3.06×10⁴ lg^-1h^-1. Based on the inactivation rate constant, and a growth rate constant determined in a previous study, the critical concentration of free chlorine was found to be 0.08 mg Cl₂/l. The critical concentration is a value below which growth rates dominate over inactivation.The authors are with the Technical University of Denmark, IMT, CDC, Build. 208, DK-2800 Lyngby, Denmark     

Combining chlorination and chloramination processes for the inhibition of biofilm formation in drinking surface water system models

AIMS: The aim of this study was to investigate the inhibition of biofilm formation on stainless steel (SS) and galvanized mild steel (MS) in chlorine (AFC(1)) and chlorine-monochloramine treated waters (AFC(2)M). METHODS AND RESULTS: Disinfection was carried out using 2.5 mg l(-1) free chlorine followed by 1.5 mg l(-1) monochloramine, with non-disinfected water used as control water. Results of the standard spread plate procedure, DAPI epifluorescence microscopy and scanning electron microscopy revealed bacterial colonization of SS and MS exposed to non-disinfected and chlorinated waters between 24 and 720 h, while no bacterial adhesion was detected on SS and MS exposed to AFC(2)M between 48 and 504 h. CONCLUSIONS: The inability of bacteria to grow on SS and MS was observed only when 0.35 mg l(-1) residual monochloramine was maintained throughout the system. SIGNIFICANCE AND IMPACT OF THE STUDY: This demonstrates the inability of chlorine alone to inhibit bacterial growth and suggests a combination of chlorine and monochloramine as a more effective treatment for drinking water, especially for rural communities with very poor source waters in Africa.     

A note on legionellas in whirlpools

Water samples from 52 whirlpools (jacuzzi), water temperature 35-40 degrees C, and from 50 swimming pools, water temperature 8-30 degrees C, were investigated for the presence of Legionella pneumophila. This was isolated from 11 of 28 whirlpools with free available chlorine less than 0.3 mg/l. No legionellas were detected in 23 whirlpools with free available chlorine over 0.3 mg/l. Legionella pneumophila was found in two swimming pools. The results indicate that 0.3 mg/l of free available chlorine is sufficient to eliminate legionellas from whirlpools.     

Effect of chlorine treatment on infectivity of hepatitis A virus

This study examined the effect of chlorine treatment on the infectivity of hepatitis A virus (HAV). Prodromal chimpanzee feces, shown to induce hepatitis in marmosets (Saguinus sp.), was clarified, and the virus was precipitated with 7% polyethylene glycol 6000, harvested, and resuspended. The suspension was layered onto 5 to 30% linear sucrose gradients and centrifuged; the fractions containing HAV were dialyzed, and a 1:500,000 dilution of this preparation induced hepatitis and seroconversion in 2 of 4 marmosets. A 1:50 dilution of this preparation served as inoculum. Untreated inoculum induced overt hepatitis and seroconversion in 100% (5 of 5) of marmosets inoculated intramuscularly. Inoculum treated for various periods (15, 30, or 60 min) with 0.5, 1.0, or 1.5 mg of free residual chlorine per liter induced hepatitis in 14% (2 of 14), 8% (1 of 12), and 10% (1 of 10) of marmosets, respectively, and induced seroconversion in 29, 33, and 10% of the animals. Inoculum treated with 2.0 or 2.5 mg of free residual chlorine per liter was not infectious in marmosets as determined by absence of hepatitis and seroconversion in the 13 animals tested. Thus, treatment levels of 0.5 to 1.5 mg of free residual chlorine per liter inactivated most but not all HAV in the preparation, whereas concentrations of 2.0 and 2.5 mg of free residual chlorine per liter destroyed the infectivity completely. These results suggest that HAV is somewhat more resistant to chlorine than are other enteroviruses.     

Effect of chlorine treatment on infectivity of hepatitis A virus.

This study examined the effect of chlorine treatment on the infectivity of hepatitis A virus (HAV). Prodromal chimpanzee feces, shown to induce hepatitis in marmosets (Saguinus sp.), was clarified, and the virus was precipitated with 7% polyethylene glycol 6000, harvested, and resuspended. The suspension was layered onto 5 to 30% linear sucrose gradients and centrifuged; the fractions containing HAV were dialyzed, and a 1:500,000 dilution of this preparation induced hepatitis and seroconversion in 2 of 4 marmosets. A 1:50 dilution of this preparation served as inoculum. Untreated inoculum induced overt hepatitis and seroconversion in 100% (5 of 5) of marmosets inoculated intramuscularly. Inoculum treated for various periods (15, 30, or 60 min) with 0.5, 1.0, or 1.5 mg of free residual chlorine per liter induced hepatitis in 14% (2 of 14), 8% (1 of 12), and 10% (1 of 10) of marmosets, respectively, and induced seroconversion in 29, 33, and 10% of the animals. Inoculum treated with 2.0 or 2.5 mg of free residual chlorine per liter was not infectious in marmosets as determined by absence of hepatitis and seroconversion in the 13 animals tested. Thus, treatment levels of 0.5 to 1.5 mg of free residual chlorine per liter inactivated most but not all HAV in the preparation, whereas concentrations of 2.0 and 2.5 mg of free residual chlorine per liter destroyed the infectivity completely. These results suggest that HAV is somewhat more resistant to chlorine than are other enteroviruses.     

Predicting the distribution of invasive crayfish (Pacifastacus leniusculus) in a Kusiro Moor marsh (Japan) using classification and regression trees

Summer distributions of the invasive signal crayfish (Pacifastacus leniusculus) were investigated in relation to physicochemistry in a Kusiro Moor marsh and its inflows and outflows in northern Japan. Maximum crayfish abundance and biomass were 1.04 individuals/m² and 3.56 g dry mass (DM)/m² in littoral marsh habitats, and 5.84 individuals/m² and 13.48 g DM/m² in stream habitats. Classification tree analysis was used to predict crayfish occurrence at 102 sites from all habitats (i.e. littoral marsh, pelagic marsh and stream) while regression tree analyses were used to predict crayfish abundance at littoral marsh and stream sites separately. The classification tree showed that crayfish occurrence was primarily determined by undercut bank volume regardless of habitat identity. When undercut bank volume was <0.0054 m³, crayfish were predicted to be absent at marsh sites, but expected to occur at stream sites where pH and water temperature exceeded 6.5 and 14.3°C, respectively. The regression tree using only littoral marsh sites showed that undercut bank volume, followed by dissolved oxygen level, determined the splits of the tree. Crayfish abundance was highest when undercut bank volume was >0.61 m³, and moderately high when dissolved oxygen was >9.09 mg/l and undercut bank volume was <0.61 m³. On the other hand, the regression tree using only stream sites showed that water temperature was the major predictor that determined the splits. We discuss the roles of physicochemical factors as limiting factors of the distribution pattern of the invasive crayfish.     

Toxicity of diflubenzuron, pyriproxyfen, imidacloprid and diafenthiuron to the predatory bugOrius laevigatus (Het.: Anthocoridae)

The susceptibility of the predatory bugOrius laevigatus (Fieber) to the insect growth regulators diflubenzuron, pyriproxyfen, the nitroguanidine insecticide imidacloprid and the thiourea compound diafenthiuron was investigated in the laboratory. Fifth-instar nymphs were exposed to formulated materials of each compound and adults were exposed to formulated materials of diafenthiuron and imidacloprid. In each case, exposure via ingestion and residual contact was tested. Pyriproxyfen was harmless toO. laevigatus nymphs by both ways of exposure. The respective LC₅₀-values of diflubenzuron via ingestion and residual contact were 229.9 and 391.1 mg a.i./l. Diafenthiuron did not cause significant mortality to fifth-instar nymphs and adults via ingestion but was toxic by residual contact with LC₅₀-values of 329.4 mg a.i./l and 125.9 mg a.i./l for nymphs and adults respectively. Imidacloprid proved to be the most toxic compound with LC₅₀-values of 1.1 and 0.04 mg a.i./l for nymphs and 2.1 and 0.3 mg a.i./l for adults, via ingestion and residual contact, respectively. The results suggest that use of pyriproxyfen in an integrated pest management programme will not cause any problems but that imidacloprid, and to a lesser extent, also diflubenzuron and diafenthiuron could be harmful to the predator.     

Resorption versus secretion in the rat epididymis

Micropuncture samples were taken from the rete testis, caput epididymidis and cauda epididymidis of anaesthetized adult rats and assayed for total protein, sodium and potassium concentrations. Intraluminal sperm concentrations were determined and used to calculate the amount of fluid resorbed from the efferent duct and epididymal lumen. It was demonstrated that large amounts of protein (30.2 mg/ml cauda volume) and sodium (241.8 mequiv./l) and smaller amounts of potassium (19.4 mequiv./l) are resorbed from the rat epididymal lumen between the caput and corpus epididymidis. This occurs despite increases in intraluminal concentrations of protein (from 22 to 28 mg/ml) and potassium (from 16 to 50 mequiv./l). Resorption is an important aspect of epididymal control of the intraluminal environment.     

Preliminary studies on continuous chromium(VI) biological removal from wastewater by anaerobic-aerobic activated sludge process

The long-term continuous chromium(VI) removal from synthetic wastewater affected by influent hexavalent chromium (Cr(VI)) and glucose concentrations were studied with an anaerobic-aerobic activated sludge process. It was observed that before activated sludge was acclimated, the chromium in the effluent increased immediately as the influent chromium increased. However, both Cr(VI) and total chromium (TCr) in the effluent significantly decreased after acclimation. In the acclimated activated sludge, the chromium removal efficiency was 100% Cr(VI) and 98.56% TCr at influent Cr(VI) levels of 20 mg/day, 100% Cr(VI) and 98.92% TCr at influent Cr(VI) levels of 40 mg/day, and 98.64% Cr(VI) and 97.16% TCr at influent Cr(VI) levels of 60 mg/day. The corresponding effluent Cr(VI) and TCr concentrations were 0 and 0.012 mg/l, 0 and 0.018 mg/l, and 0.034 mg/l and 0.071 mg/l, respectively. When the influent glucose increased from 1125 to 1500 mg/l at influent Cr(VI) dosage of 60 mg/day, the Cr(VI) and TCr removal efficiency with the acclimated activated sludge improved from 98.64% and 97.16% to 100% and 98.48%, respectively, and the chromium concentration in the effluent decreased from 0.034 mg/l of Cr(VI) and 0.071 mg/l of TCr to 0 (Cr(VI)) and 0.038 mg/l (TCr). The effluent COD and turbidity was around 40 mg/l and 0, respectively, after the activated sludge was acclimated. Further studies showed that after the activated sludge was acclimated, its specific dehydrogenases activity (SDA) and protein contents increased. The SDA and protein increased respectively 15% and 10% when influent Cr(VI) increased from 20 to 60 mg/day.