Distribution and toxicity of sediment-associated pesticides in agriculture-dominated water bodies of California's Central Valley

The agricultural industry and urban pesticide users are increasingly relying upon pyrethroid insecticides and shifting to more potent members of the class, yet little information is available on residues of these substances in aquatic systems under conditions of actual use. Seventy sediment samples were collected over a 10-county area in the agriculture-dominated Central Valley of California, with most sites located in irrigation canals and small creeks dominated by agricultural effluent. The sediments were analyzed for 26 pesticides including five pyrethroids, 20 organochlorines, and one organophosphate. Ten-day sediment toxicity tests were conducted using the amphipod Hyalella azteca and, for some samples, the midge Chironomus tentans. Forty-two percent of the locations sampled caused significant mortality to one test species on at least one occasion. Fourteen percent of the sites (two creeks and four irrigation canals) showed extreme toxicity (>80% mortality) on at least one occasion. Pyrethroid pesticides were detected in 75% of the sediment samples, with permethrin detected most frequently, followed by esfenvalerate > bifenthrin > lambda-cyhalothrin. Based on a toxicity unit analysis, measured pyrethroid concentrations were sufficiently high to have contributed to the toxicity in 40% of samples toxic to C. tentans and nearly 70% of samples toxic to H. azteca. Organochlorine compounds (endrin, endosulfan) may have contributed to the toxicity at a few other sites. This study provides one of the first geographically broad assessments of pyrethroids in areas highly affected by agriculture, and it suggests there is a greater need to examine sediment-associated pesticide residues and their potential for uptake by and toxicity to benthic organisms.     

Pyrethroid insecticides and sediment toxicity in urban creeks from California and Tennessee

Pyrethroid pesticides have replaced organophosphates for many urban applications, including structural pest control, landscape maintenance, and residential home and garden use. This study was intended to determine if pyrethroids are detectable and widespread in diverse urban systems and if concentrations are high enough to cause associated aquatic toxicity. Urban creeks in California and Tennessee were tested on up to four occasions for pesticide residues in sediments, and aquatic toxicity was determined by acute toxicity tests using the amphipod, Hyalella azteca. In California, 12 of the 15 creeks tested were toxic on at least one sampling occasion, and sediment pyrethroid concentrations were sufficient to explain the observed toxicity in most cases. The pyrethroid bifenthrin, due to its high concentrations and relative toxicity as compared to other pyrethroids, was likely responsible for the majority of the toxicity at most sites. Cypermethrin, cyfluthrin, deltamethrin, and lambda-cyhalothrin also contributed to toxicity at some locations. The source of cypermethrin and deltamethrin was probably almost entirely structural pest control by professional applicators. Bifenthrin, cyfluthrin, and lambda-cyhalothrin may have originated either from professional structural pest control or from lawn and garden care by homeowners. None of the sediments collected from the 12 Tennessee creeks were toxic, and pyrethroids were rarely detectable. Regional differences between Tennessee and California are possibly attributable to climate, differences in types of residential development, and pesticide use practices.     

Aquatic toxicity due to residential use of pyrethroid insecticides

Pyrethroids are the active ingredients in most insecticides available to consumers for residential use in the United States. Yet despite their dominance in the marketplace, there has been no attempt to analyze for most of these compounds in watercourses draining residential areas. Roseville, California was selected as a typical suburban development, and several creeks that drain subdivisions of single-family homes were examined. Nearly all creek sediments collected caused toxicity in laboratory exposures to an aquatic species, the amphipod Hyalella azteca, and about half the samples caused nearly complete mortality. This same species was also found as a resident in the system, but its presence was limited to areas where residential influence was least. The pyrethroid bifenthrin is implicated as the primary cause of the toxicity, with additional contributions to toxicity from the pyrethroids cyfluthrin and cypermethrin. The dominant sources of these pyrethroids are structural pest control by professional applicators and/ or homeowner use of insecticides, particularly lawn care products. The suburbs of Roseville are unlikely to be unique, and similar sediment quality degradation is likely in other suburban areas, particularly in dry regions where landscape irrigation can dominate seasonal flow in some water bodies.     

Occurrence and potential sources of pyrethroid insecticides in stream sediments from seven U.S. metropolitan areas

A nationally consistent approach was used to assess the occurrence and potential sources of pyrethroid insecticides in stream bed sediments from seven metropolitan areas across the United States. One or more pyrethroids were detected in almost half of the samples, with bifenthrin detected the most frequently (41%) and in each metropolitan area. Cyhalothrin, cypermethrin, permethrin, and resmethrin were detected much less frequently. Pyrethroid concentrations and Hyalella azteca mortality in 28-d tests were lower than in most urban stream studies. Log-transformed total pyrethroid toxic units (TUs) were significantly correlated with survival and bifenthrin was likely responsible for the majority of the observed toxicity. Sampling sites spanned a wide range of urbanization and log-transformed total pyrethroid concentrations were significantly correlated with urban land use. Dallas/Fort Worth had the highest pyrethroid detection frequency (89%), the greatest number of pyrethroids (4), and some of the highest concentrations. Salt Lake City had a similar percentage of detections but only bifenthrin was detected and at lower concentrations. The variation in pyrethroid concentrations among metropolitan areas suggests regional differences in pyrethroid use and transport processes. This study shows that pyrethroids commonly occur in urban stream sediments and may be contributing to sediment toxicity across the country.     

Aquatic effects of aerial spraying for mosquito control over an urban area

In an effort to combat West Nile Virus, planes dispersed insecticide over Sacramento, CA, treating nearly 50,000 hectares with pyrethrins and the synergist piperonyl butoxide (PBO). Widespread dispersal of insecticide over a metropolitan area, coupled with extensive pretreatment data on the area's urban creeks, provided a unique opportunity to study effects of mosquito control agents on aquatic habitats within an urban setting. There was no evidence of aquatic toxicity from the two active ingredients in the product applied. However, PBO concentrations were high enough to enhance toxicity of pyrethroids already existing in creek sediments from general urban pesticide use. PBO concentrations of 2-4 microg/L were high enough to nearly double the toxicity of sediments to the amphipod Hyalella azteca. Though the increase in toxicity was modest, it was unexpected to find environmental synergy at all. Risk assessments for mosquito control agents have focused on the active ingredients but have failed to recognize the potential for interactions with pesticides previously existing in the environment, which in this case appeared to represent a risk to aquatic life greater than that of the active ingredients themselves.     

Identifying the causes of sediment-associated toxicity in urban waterways of the Pearl River Delta, China

Twenty-one sediments collected in urban waterways of the Pearl River Delta (PRD) in China were screened for acute toxicity using Chironomus dilutus in addition to being examined for potential contributors of sediment toxicity, including 19 organochlorine, five organophosphate, and nine pyrethroid insecticides, 28 polycyclic aromatic hydrocarbons, 27 polychlorinated biphenyls, 15 polybrominated diphenyl ethers, 12 metals, and ammonia. Fifteen of the 21 sediments exhibited acute toxicity to C. dilutus, with 33% of the samples exhibiting 100% mortality. This is one of the first studies in China which directly correlates a broad range of sediment-associated contaminants to ecological effects measured by bioassays. A toxic unit approach showed that pyrethroids contributed most to the observed toxicity; as cypermethrin alone was predicted to cause significant mortality in about half of the sites. Specific toxicity identification evaluation analysis confirmed pyrethroid toxicity. Other contaminants may also be supplemental contributors at a few sites. The current study suggests that pyrethroids are the principal cause of contamination and that future risk assessment and mitigation efforts in this area should focus primarily on pyrethroids but should not disregard other contaminants as potential risk is evident.     

Effect of dissolved organic carbon on sorption of pyrethroids to sediments

Despite their strong hydrophobicity, recent studies showed widespread occurrence of pyrethroid in downstream surface waters bodies. In this work, the effect of dissolved organic carbon (DOC) on the sorption and desorption of pyrethroids in sediment was evaluated to understand the role of DOC in facilitating pyrethroid transport. Presence of DOC from three sources at 38 ± 2 mg L?1 in the aqueous phase decreased pesticide sorption to a sediment by 1.7 to 38.9 times and increased their desorption by 1.2 to 41.4 times. The effect on pyrethroid sorption to the sediment was linear. In addition, interactions between DOC and pyrethroids, when taking place prior to the contact with sediment, decreased sorption of some pyrethroids even further, implying that DOC-pyrethroid complexs were relatively stable in solution. DOC sources with higher contents of carboxylic and phenolic groups were found to have a higher potential to associate with pyrethroids. The DOC-water partition coefficients (K(DOC)) obtained by solid-phase microextraction measurement were significantly correlated (P < 0.01) with K(d) values measured for the sediment. These results provide evidence that DOC increases the distribution of pyrethroids from the sediment to the solution phase and plays an important role in mobilizing pyrethroids in runoff and surface streams.     

枸杞中拟除虫菊酯类农药残留水平及累积暴露评估

目的检测枸杞中的农药残留水平,并对拟除虫菊酯类农药的累积暴露风险进行评估,为是否需要制定枸杞中最大残留限量提供依据。方法收集40份不同来源不同产地的枸杞样品,对332种农药残留进行测定,其中包括15种拟除虫菊酯类农药。采用危害指数(HI)法对通过枸杞摄入拟除虫菊酯类农药产生的急性和慢性累积风险进行评估。结果通过方法学验证得出此方法适用于枸杞中拟除虫菊酯类农药的测定。结果显示4种拟除虫菊酯农药检出率均高于25%,分别为氯氰菊酯、氰戊菊酯、甲氰菊酯和氯氟氰菊酯。采用HI法进行累积暴露评估,得出拟除虫菊酯类农药的急性和慢性危害指数分别为0.497和0.016。结论枸杞在拟除虫菊酯类农药的累积摄入风险虽然较小,但由于其检出率较高,应持续保持对样品的监控,并考虑制定相应的最大残留限量值。     

Determination of Pyrethroid Insecticides in Crude and Canned Vegetable Samples by Supercritical Fluid Chromatography

A supercritical fluid chromatography method for the determination of seven pyrethroid insecticides (allethrin, resmethrin, phenothrin, permethrin, tetramethrin, cypermethrin, deltamethrin) and one of their common metabolites, phenoxybenzyl alcohol, in whole and peeled potatoes and mixed vegetables was developed. The efficiencies of the two extraction techniques, supercritical fluid extraction and microwave-assisted extraction, for the extraction of pyrethroids from vegetable samples were also compared. The retention times of various pyrethroids ranged from 8.4 to 22.9 min, while all of the peaks were well-resolved and distinctly identified. The limits of detection of pyrethroid insecticides ranged between 0.31 and 0.54 ppm, whereas the limits of detection of phenoxybenzyl alcohol was 0.62 ppm. The recoveries of pyrethroid insecticides from whole potatoes, peeled potatoes, and mixed vegetables ranged as 93.83–99.8%, 92.3–105.8%, and 93.67–102.7%, respectively, with the use of supercritical fluid extraction. The corresponding recovery ranges while using microwave-assisted extraction were 94.2–102%, 96.6–101.2%, and 96–103.2%. These findings suggested that supercritical fluid chromatography was a sensitive and rapid technique for the analysis of pyrethroids in complex matrices, such as vegetables, fruits, and other agricultural products.     

水果中26种有机氯及拟除虫菊酯农药残留GC-ECD测定

利用气相色谱(GC)建立了水果中26种有机氯农药同时测定的方法。试样中农药残留在超声波振荡条件下用乙腈提取,经PSA、C18净化,GC-电子捕获检测器(ECD)检测。方法的检出限为0.001～0.005mg/kg,回收率为84.5%～104.5%,相对标准偏差为1.1%～6.7%。该方法的灵敏度、准确度和精密度均符合农药残留测定的技术要求。     

Insecticide resistance and synergism in Brazilian populations of Sitophilus zeamais (Coleoptera: Curculionidae)

Insecticide resistance to three organophosphate (chlorpyrifos-methyl, malathion and pirimiphos-methyl) and three pyrethroid (cypermethrin, deltamethrin and permethrin) insecticides was surveyed in nine field-collected populations of the maize weevil from six Brazilian States and contrasted with a standard susceptible population and a DDT and pyrethroid resistant population. Bioassay tests were carried out in glass vials (20 ml) impregnated with dried insecticide residue and the resistant populations were recognized by using discriminating concentrations established from LC₉₅s estimated for a standard susceptible population. Four field-collected populations had reduced susceptibility to cypermethrin (64-77% mortality) and to chlorpyrifos-methyl (76-77% mortality). All populations were susceptible to malathion and pirimiphos-methyl and only the DDT and pyrethroid resistant check showed resistance to all pyrethroids. Concentration-mortality curves showed non-significant levels of insecticide resistance in the field-collected populations and moderate (10-100-fold) to very high levels (>1000-fold) of resistance to pyrethroids in the DDT and pyrethroid resistant population. Results from insecticide bioassays with synergists (diethyl maleate, piperonyl butoxide, and triphenyl phosphate) suggested a major involvement of esterase as the cypermethrin resistance mechanism, while no clear result was obtained for chlorpyrifos-methyl. Synergism results provide support for target site resistance to pyrethroids in the DDT and pyrethroid resistant population used in this study.     

Toxicity of amorphous silicas and silica-pyrethroid mixtures to Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae)

The toxicity of some pyrogenic and precipitated amorphous silicas to Tribolium castaneum (Herbst) was assessed in admixture with wheat grain. A good correlation (r² = 0.82, n = 7) was found between the 48 h LC50 of the toxic dusts and their adsorption capacity for a saturated hydrocarbon oil. The pyrogenic silicas Cab-O-Sil M5 and Aerosil R972 were used as carriers to prepare dust formulations of permethrin, cypermethrin and deltamethrin at various concentrations, and 48 h (Cab-O-Sil M5 and Aerosil R972) and 168 h (Cab-O-Sil M5) LC50s were determined using the same bioassay system. Low and intermediate concentrations of the pyrethroids substantially reduced the 48 h LC50s of these silicas while high concentrations antagonised their toxic action due to knockdown effects. The 168 h LC50s of Cab-O-Sil/permethrin formulations were approx 15 and 150 × lower than those of talc/permethrin formulations at 2% w/w and 0.1% w/w respectively. The 168 h LC50 of the Cab-O-Sil/cypermethrin formulation was comparable with that of the talc/cypermethrin formulation, and that of the Cab-O-Sil/deltamethrin formulation was about 5 × less than a talc/deltamethrin formulation, at 2 and 0.2% w/w pyrethroid respectively on the dust carrier.     

Time-dependent toxicity of fluoranthene to freshwater invertebrates and the role of biotransformation on lethal body residues

The time-dependent toxicity of fluoranthene was examined for Hyalella azteca, Chironomus tentans, and Diporeia spp. C. tentans appeared to be the most sensitive species, and Diporeia was the least sensitive. Incipient LC50 values, the concentration at which the LC50 reaches an asymptote and does not change with increasing duration of exposure, for H. azteca and C. tentans were approximately 60 and 40 microg x L(-1), respectively. Incipient levels were not reached for Diporeia; however, the 28-d LC50 concentration was 95.5 microg x L(-1). There was a temporal relationship with respect to lethal body residues for each of the test species. For H. azteca, the LR50, the median lethal residue at an identified exposure time required to cause 50% mortality, based on total fluoranthene equivalents (parent + metabolite compounds) decreased from 3.19 micromol x g(-1) at 5 d to 0.80 micromol x g(-1) at 28 d. For C. tentans, the LR50 decreased from 0.43 to 0.17 micromol x g(-1) from 2 to 10 d. The 10-d LR50 for Diporeia was 9.97 micromol x g(-1), and the 28-d value was 3.67 micromol x g(-1). The toxicokinetics are not sufficient to address the temporal changes in LR50 values. Thus, the data were fit to a Damage Assessment Model that also accounts for toxicodynamic processes. This analysis provides estimates of the incipient lethal residues for H. azteca, C. tentans, and Diporeia: 0.84, 0.21, and 3.00 micromol x g(-1), respectively. When comparing the relative sensitivity among species using lethal body residues, special attention should be given to ensure that comparisons are made at a common point in relation to exposure duration (i.e., time to steady state, Tss). When the LR50(lipid) values among the three species were compared at steady state, C. tentans is more sensitive than H. azteca and Diporeia spp.; however, there are no significant differences between the amphipod species. The greater sensitivity of C. tentans to fluoranthene as compared to the amphipods may be due, in part, to a potential toxic metabolite.     

基于体外模拟消化模型评价刺参中3种拟除虫菊酯的生物可给性

目的 评估刺参中拟除虫菊酯的生物可给性,为海产品中农药残留的监管提供参考。方法 基于胃相动态pH的体外模拟消化模型,采用气相色谱法分析加工前后刺参(Stichopus japonicas)中3种拟除虫菊酯(甲氰菊酯、氯氰菊酯和溴氰菊酯)的含量。结果 加工组与原料组相比,经过胃相消化后,3种菊酯类含量均显著降低(P<0.05);不管是原料组还是加工组,与消化前相比,经过肠道模拟消化后,3种菊酯浓度均显著降低(P<0.05);原料组中甲氰菊酯,氯氰菊酯和溴氰菊酯的生物可给性分别为63.09%,86.67%和71.83%,除了溴氰菊酯,加工后另外两种菊酯类生物可给性均低于原料组。结论 本研究结果表明刺参中3种拟除虫菊酯经模拟消化后均不能从食物基质中完全释放,此外加热处理可以在一定程度上降低刺参中拟除虫菊酯的生物可给性。     

Effects of sediment characteristics on the toxicity of chromium(III) and chromium(VI) to the amphipod, Hyalella azteca

We evaluated the influence of sediment characteristics, acid-volatile sulfide (AVS) and organic matter (OM), on the toxicity of chromium (Cr) in freshwater sediments. We conducted chronic (28-42-d) toxicitytests with the amphipod Hyalella azteca exposed to Cr(VI) and Cr(III) in water and in spiked sediments. Waterborne Cr(VI) caused reduced survival of amphipods with a median lethal concentration (LC50) of 40 microg/L. Cr(VI) spiked into test sediments with differing levels of AVS resulted in graded decreases in AVS and sediment OM. Only Cr(VI)-spiked sediments with low AVS concentrations (< 1micromol/g) caused significant amphipod mortality. Waterborne Cr(III) concentrations near solubility limits caused decreased survival of amphipods at pH 7 and pH 8 but not at pH 6. Sediments spiked with high levels of Cr(III) did not affect amphipod survival but had minor effects on growth and inconsistent effects on reproduction. Pore waters of some Cr(III)-spiked sediments contained measurable concentrations of Cr(VI), but observed toxic effects did not correspond closely to Cr concentrations in sediment or pore waters. Our results indicate that risks of Cr toxicity are low in freshwater sediments containing substantial concentrations of AVS.     

Comparison of five methods for measuring sediment toxicity of hydrophobic contaminants

Sediment toxicity from hydrophobic organic compounds (HOCs) is complicated by chemical partitioning among multiple phases and sediment-specific bioavailability. In this study, we used three hydrophobic pyrethroid insecticides as test compounds and derived 10-d median lethal concentrations (LC50s) for Chironomus tentans in three different sediments. The LC50s were expressed using HOC concentrations on a bulk sediment basis (C(S)), organic carbon (OC)-normalized sediment basis (C(S-OC)), porewater basis (C(PW)), dissolved organic carbon (DOC)-normalized porewater basis (C(PW-DOC)), and freely dissolved porewater basis (C(free)). The bulk phase C(S) and C(PW) yielded highly variable LC50s across sediment types, whereas the use of normalized concentrations C(S-OC) and C(PW-DOC) generally reduced variability due to sediment type but not that due to aging. In contrast, LC50s based on C(free) were essentially independent of sediment conditions. The sediment pore water samples contained approximately 20-90 mg L(-1) DOC, and the C(free) expressed as a percentage of the total bulk pore water concentration ranged from 9 to 28% for fenpropathrin (mean = 19%), 8 to 18% for bifenthrin (mean = 13%), and 3 to 8% for cyfluthrin (mean = 6%) across the different sediments. These results indicate thatthe use of C(free) reduces uncertainties caused by sediment variables such as OC properties and aging effects.     

Selective treatments,including combinations of pyrethroid and organophosphorus insecticides. For control of stored product Coleoptera at two temperatures

Selective treatments, appropriate to simulated storage conditions, reinfestation pressures and species or strains of insects present in the wheat, required less insecticide than any “blanket” or all-purpose treatment. In particular, the pyrethroids bioresmethrin, synergized bioresmethrin or synergized pyrethrins were especially effective where Rhyzopertha dominica and especially a strain resistant to organophosphorus insecticides, was the only species present. Conversly, the organophosphorus insecticides dichlorvos, malathion, fenitrothion and pirimiphos-methyl were especially effective against Tribolium and Sitophilus species. There was no antagonism between any of the pyrethroids and any of the organophosphorus insecticides when they were applied in combination, in that the period of protection given by combinations was the period of protection given by the pyrethroid component against R. dominica and the organophosphorus component against Tribolium and Sitophilus species. As an example of the data required for selective treatments, bioassays were conducted at 20 and 30°C and it was found that the net effect of the lower temperature in reducing toxicity but increasing persistence was to increase the period of protection given by insecticides in cooled storages. It is suggested that the amounts of insecticides applied to grain could be considerably reduced if more consideration was given to grain conditions, if reinfestation pressures were more carefully examined and if less attention were given to surviving adults under conditions where progeny do not develop.     

Predicting bioavailability of sediment polycyclic aromatic hydrocarbons to Hyalella azteca using equilibrium partitioning, supercritical fluid extraction, and pore water concentrations

Polycyclic aromatic hydrocarbon (PAH) bioavailability to Hyalella azteca was determined in 97 sediments from six former manufactured-gas plants and two aluminum smelter sites. Measurements of Soxhlet extractable, rapidly released based on mild supercritical fluid extraction, and pore water dissolved concentrations of 18 parent and 16 groups of alkyl PAHs (PAH34) were used to predict 28 daysurvival based on equilibrium partitioning and hydrocarbon narcosis models. Total PAH concentrations had little relationship to toxicity. Amphipods survived in sediments with PAH34 concentrations as high as 2990 microg/g, while sediments as low as 2.4 microg/g of PAH34 resulted in significant mortality. Equilibrium partitioning using either total extractable or rapidly released concentrations significantly improved predictions. However, pore water PAH34 concentrations were best for predicting amphipod survival and correctly classified toxic and nontoxic sediment samples with an overall model efficiency of 90%. Alkyl PAHs accounted for 80% of the toxicity, demonstrating that careful measurement of the 16 alkyl clusters in pore water is required. Regression analysis of the pore water PAH34 data from 97 field sediments against amphipod survival resulted in a mean 50% lethal residue value of 33 micromol/g of lipid, consistent with 32 micromol/g of lipid for fluoranthene determined by others in controlled laboratory conditions, thus demonstrating the applicability of EPA's hydrocarbon narcosis model when using pore water PAH34 concentrations.     

Survival of Listeria monocytogenes in a food colorant derived from red beets

Three commercial lots of the red beet colorant were inoculated to contain circa 10(3) to 10(7) Listeria monocytogenes strains California, V7, or Scott A per milliliter and stored for 56 d at 7 degrees C. McBride listeria agar was used to determine numbers of survivors. Selected colonies thought to be L. monocytogenes were confirmed biochemically. When necessary, samples were tested by cold enrichment (up to 8 weeks). Samples of colorant initially containing 10(3) to 10(4) strain California/ml were always free of the pathogen after 56 d, and sometimes after 42 d. Samples with high initial numbers (10(5) to 10(6)/ml) were not free of the pathogen after 8 wk at 7 degrees C. Strains V7 and Scott A, regardless of size of initial population, always survived beyond 56 d. Before inoculation, all test samples of colorant were free of L. monocytogenes (direct plating or cold enrichment).     

Inhibition of growth of nonproteolytic Clostridium botulinum type B in sous vide cooked meat products is achieved by using thermal processing but not nisin

The safety of refrigerated processed foods of extended durability (REPFEDs) with respect to nonproteolytic Clostridium botulinum is under continuous evaluation. In the present study, mild (P7.0(85.0) values 0 to 2 min [P, pasteurization value; z-value 7.0 degrees C; reference temperature 85.0 degrees C]) and increased (P7.0(85.0) values 67 to 515 min) heat treatments were evaluated in relation to survival of nonproteolytic C. botulinum type B spores in sous vide processed ground beef and pork cubes. The use of two concentrations of nisin in inhibition of growth and toxin production by nonproteolytic C. botulinum in the same products was also evaluated. A total of 96 samples were heat processed and analyzed for C. botulinum by BoNT/B gene-specific polmerase chain reaction and for botulinum toxin by a mouse bioassay after storage of 14 to 28 days at 4 and 8 degrees C. Predictably, after mild processing all samples of both products showed botulinal growth, and one ground beef sample became toxic at 8 degrees C. The increased heat processing, equivalent to 67 min at 85 degrees C. resulted in growth but not toxin production of C. botulinum in one ground beef sample in 21 days at 8 degrees C: in the pork cube samples no growth was detected. The increased heating of both products resulted in higher sensory quality than the milder heat treatment. Nisin did not inhibit the growth of nonproteolytic C. botulinum in either product; growth was detected in both products at 4 and 8 degrees C, and ground beef became toxic with all nisin levels within 21 to 28 days at 8 degrees C. Aerobic and lactic acid bacterial counts were reduced by the addition of nisin at 4 degrees C. The study demonstrates that the mild processing temperatures commonly employed in sous vide technology do not eliminate nonproteolytic C. botulinum type B spores. The intensity of each heat treatment needs to be carefully evaluated individually for each product to ensure product safety in relation to nonproteolytic C. botulinum.