Effects of glutaraldehyde and povidone-iodine on apoptosis of grass carp liver and hepatocytes.

Since disinfectants are used all over the world to treat illnesses in people and other animals, they pose a major risk to human health. The comprehensive effects of disinfectant treatments on fish liver, especially the impacts on oxidative stress, toxicological effects, transcriptome profiles, and apoptosis, have not yet been fully analyzed. In the current investigation, healthy grass carp were exposed to 80 µg/L glutaraldehyde or 50 µg/L povidone-iodine for 30 days. First, the findings of enzyme activity tests demonstrated that the administration of glutaraldehyde could considerably increase oxidative stress by lowering T-SOD, CAT, and GPx and raising MDA. Furthermore, KEGG research revealed that exposure to glutaraldehyde and povidone-iodine stimulated the PPAR signal pathway. To further elucidate the transcriptome results, the relative expressions of related DEGs in the PPAR signal pathway were verified. Glutaraldehyde induced apoptosis in liver tissue of grass carp; however, it activated cytotoxicity and apoptosis in grass carp hepatocytes when exposed to glutaraldehyde or povidone-iodine. According to the current study, disinfectants can cause the impairment of the immune system, oxidative stress, and attenuation of the PPAR signal pathway in the liver of grass carp, making them detrimental as dietary supplements for grass carp, particularly in the aquaculture sector.

Subacute Pulmonary Toxicity of Glutaraldehyde Aerosols in a Human In Vitro Airway Tissue Model.

Glutaraldehyde (GA) has been cleared by the Center for Devices and Radiological Health (CDRH) of the Food and Drug Administration (FDA) as a high-level disinfectant for disinfecting heat-sensitive medical equipment in hospitals and healthcare facilities. Inhalation exposure to GA is known to cause respiratory irritation and sensitization in animals and humans. To reproduce some of the known in vivo effects elicited by GA, we used a liquid aerosol exposure system and evaluated the tissue responses in a human in vitro airway epithelial tissue model. The cultures were treated at the air interface with various concentrations of GA aerosols on five consecutive days and changes in tissue function and structure were evaluated at select timepoints during the treatment phase and after a 7-day recovery period. Exposure to GA aerosols caused oxidative stress, inhibition of ciliary beating frequency, aberrant mucin production, and disturbance of cytokine and matrix metalloproteinase secretion, as well as morphological transformation. Some effects, such as those on goblet cells and ciliated cells, persisted following the 7-day recovery period. Of note, the functional and structural disturbances observed in GA-treated cultures resemble those found in ortho-phthaldehyde (OPA)-treated cultures. Furthermore, our in vitro findings on GA toxicity partially and qualitatively mimicked those reported in the animal and human survey studies. Taken together, observations from this study demonstrate that the human air-liquid-interface (ALI) airway tissue model, integrated with an in vitro exposure system that simulates human inhalation exposure, could be used for in vitro-based human hazard identification and the risk characterization of aerosolized chemicals.

Impact of small fractions of abnormal erythrocytes on blood rheology.

Red blood cell (RBC) populations are inherently heterogeneous, given mature RBC lack the transcriptional machinery to re-synthesize proteins affected during in vivo aging. Clearance of older, less functional cells thus aids in maintaining consistent hemorheological properties. Scenarios occur, however, where portions of mechanically impaired RBC are re-introduced into blood (e.g., damaged from circulatory support, blood transfusion) and may alter whole blood fluid behavior. Given such perturbations are associated with poor clinical outcomes, determining the tolerable level of abnormal RBC in blood is valuable. Thus, the current study aimed to define the critical threshold of blood fluid properties to re-infused physically-impaired RBC. Cell mechanics of RBC were impaired through membrane cross-linking (glutaraldehyde) or intracellular oxidation (phenazine methosulfate). Mechanically impaired RBC were progressively re-introduced into the native cell population. Negative alterations of cellular deformability and high shear blood viscosity were observed following additions of only 1-5% rigidified RBC. Low-shear blood viscosity was conversely decreased following addition of glutaraldehyde-treated cells; high-resolution microscopy of these mixed cell populations revealed decreased capacity to form reversible aggregates and decreased aggregate size. Mixed RBC populations, when exposed to supraphysiological shear, presented with compounded mechanical impairment. Collectively, key determinants of blood flow behavior are sensitive to mechanical perturbations in RBC, even when only 1-5% of the cell population is affected. Given this fraction is well-below the volume of rigidified RBC introduced during circulatory support or transfusion practice, it is plausible that some adverse events following surgery and/or transfusion may be related to impaired blood fluidity.

Toxicological aspects of increased use of surface and hand disinfectants in Croatia during the COVID-19 pandemic: a preliminary report.

All COVID-19 prevention strategies include regular use of surface disinfectants and hand sanitisers. As these measures took hold in Croatia, the Croatian Poison Control Centre started receiving phone calls from the general public and healthcare workers, which prompted us to investigate whether the risk of suspected/symptomatic poisonings with disinfectants and sanitisers really increased. To that end we compared their frequency and characteristics in the first half of 2019 and 2020. Cases of exposures to disinfectants doubled in the first half of 2020 (41 vs 21 cases in 2019), and exposure to sanitisers increased about nine times (46 vs 5 cases in 2019). In 2020, the most common ingredients of disinfectants and sanitisers involved in poisoning incidents were hypochlorite/glutaraldehyde, and ethanol/isopropyl alcohol, respectively. Exposures to disinfectants were recorded mostly in adults (56 %) as accidental (78 %) through ingestion or inhalation (86 %). Fortunately, most callers were asymptomatic (people called for advice because they were concerned), but nearly half reported mild gastrointestinal or respiratory irritation, and in one case severe symptoms were reported (gastrointestinal corrosive injury). Reports of exposure to hand sanitisers highlighted preschool children as the most vulnerable group. Accidental exposure through ingestion dominated, but, again, only mild symptoms (gastrointestinal or eye irritation) developed in one third of the cases. These preliminary findings, however limited, confirm that increased availability and use of disinfectants and sanitisers significantly increased the risk of poisoning, particularly in preschool children through accidental ingestion of hand sanitisers. We therefore believe that epidemiological recommendations for COVID-19 prevention should include warnings informing the general public of the risks of poisoning with surface and hand disinfectants in particular.

Evaluation of the Effect of Crosslinking Method of Poly(Vinyl Alcohol) Hydrogels on Thrombogenicity.

PURPOSE: Crosslinked poly(vinyl alcohol) (PVA) is a biomaterial that can be used for multiple cardiovascular applications. The success of implanted biomaterials is contingent on the properties of the material. A crucial consideration for blood-contacting devices is their potential to incite thrombus formation, which is dependent on the material surface properties. The goal of this study was to quantify the effect of different crosslinking methods of PVA hydrogels on in vitro thrombogenicity. METHODS: PVA was manufactured using three different crosslinking methods: 30% sodium trimetaphosphate (STMP), three 24 h freeze-thaw cycles (FT), and 2% glutaraldehyde-crosslinked (GA) to produce STMP-PVA, FT-PVA and GA-PVA, respectively. Expanded polytetrafluoroethylene (ePTFE) was used as a clinical control. As markers of thrombus formation, the degree of coagulation factor (F) XII activation, fibrin formation, and platelet adhesion were measured. RESULTS: The GA-PVA material increased FXII activation in the presence of cofactors compared to vehicle and increase platelet adhesion compared to other PVA surfaces. The STMP-PVA and FT-PVA materials had equivalent degrees of FXII activation, fibrin formation and platelet adhesion. CONCLUSION: This work supports crosslinker dependent thrombogenicity of PVA hydrogels and advances our understanding of how the manufacturing of a PVA hydrogel affects its hemocompatibility.

Glutaraldehyde Cross-Linking of Oligolysines Coating DNA Origami Greatly Reduces Susceptibility to Nuclease Degradation.

DNA nanostructures (DNs) have garnered a large amount of interest as a potential therapeutic modality. However, DNs are prone to nuclease-mediated degradation and are unstable in low Mg2+ conditions; this greatly limits their utility in physiological settings. Previously, PEGylated oligolysines were found to protect DNs against low-salt denaturation and to increase nuclease resistance by up to ∼400-fold. Here we demonstrate that glutaraldehyde cross-linking of PEGylated oligolysine-coated DNs extends survival by up to another ∼250-fold to >48 h during incubation with 2600 times the physiological concentration of DNase I. DNA origami with cross-linked oligolysine coats are non-toxic and are internalized into cells more readily than non-cross-linked origami. Our strategy provides an off-the-shelf and generalizable method for protecting DNs in vivo.

Multimodal detection and analysis of a new type of advanced Heinz body-like aggregate (AHBA) and cytoskeleton deformation in human RBCs.

A new type of aggregate, formed in human red blood cells (RBCs) in response to glutaraldehyde treatment, was discovered and analyzed with the classical and advanced biomolecular imaging techniques. Advanced Heinz body-like aggregates (AHBA) formed in a single human RBC are characterized by a higher level of hemoglobin (Hb) degradation compared to typical Heinz bodies, which consist of hemichromes. The complete destruction of the porphyrin structure of Hb and the aggregation of the degraded proteins in the presence of Fe3+ ions are observed. The presence of such aggregated, highly degraded proteins inside RBCs, without cell membrane destruction, has been never reported before. For the first time the spatial differentiation of two kinds of protein mixtures inside a single RBC, with different phenylalanine (Phe) conformations, is visualized. The non-resonant Raman spectra of altered RBCs with AHBA are characterized by the presence of a strong band located at 1037 cm-1, which confirms that glutaraldehyde interacts strongly with Phe. The shape-shifting of RBCs from a biconcave disk to a spherical structure and sinking of AHBA to the bottom of the cell are observed. Results reveal that the presence of AHBA should be considered when fixing RBCs and indicate the analytical potential of Raman spectroscopy, atomic force microscopy and scanning near-field optical microscopy in AHBA detection and analysis.

Environmental electroactive consortia as reusable biosensing element for freshwater toxicity monitoring.

The development of tools to monitor water quality is mandatory in a scenario where clean water resources are decreasing. Here, the biosensing capability of an electroactive river sediment consortium was tested towards three model contaminants (glutaraldehyde, nickel(II) and chromium(III)). The proposed biosensor is a small membrane-less single chamber Microbial Fuel Cell (MFC), fabricated by 3D printing. Its semi-continuous mode of operation resulted in long-term current profile stability and reproducibility. A linear trend of response was obtained for glutaraldehyde in a concentration range of 5-1000 ppm. After the recovery of the electroactive consortium activity, the MFC-based biosensors were shown to be sensitive towards Ni(II) and Cr(III), at concentrations above 2 mg L-1. To effectively analyze biosensor response, a novel algorithm was proposed, offering advantages for the realization of energy-saving protocols for MFC-biosensor data transmission. Implementation of the device and method, from laboratory test to real environment, can offer a low cost in situ system for detection of water contaminants.

Toxicity and Metal Corrosion of Glutaraldehyde-Didecyldimethylammonium Bromide as a Disinfectant Agent.

The wide use of disinfectants has prompted resistance from the microbiome which will in turn reduce the bactericidal effect of disinfectants. Hence, glutaraldehyde (GA) and didecyldimethylammonium bromide (DDAB) were used to develop a combination disinfectant with high stability and antimicrobial effects, which was named GA-DDAB combination disinfectant (GD). The bactericidal mechanism against Escherichia coli was studied in our earlier work. In this study, we focused on GD's bactericidal efficacy in both the laboratory and environment, the genetic toxicity to mouse lymphoma L5178Y TK+/- cells, acute peroral toxicity in mice, and its metal corrosion properties with a view to providing theoretical support for developing a high-efficiency, low toxicity, and weakly corrosive disinfectant for general use.

Reversible renal glomerular dysfunction in guinea pigs exposed to glutaraldehyde-polymerized cell-free hemoglobin.

Chemically modified hemoglobin (Hb)-based oxygen carriers are promising oxygen replacement therapeutics however their potential renal effects are not fully understood. Using a guinea pig exchange transfusion model, we examined the effects of glutaraldehyde-polymerized bovine hemoglobin (HbG) on the permeability and integrity of the glomerular filtration barrier (GFB), which is comprised of podocytes, fenestrated endothelium, and the glomerular basement membrane. HbG induced marked proteinuria characterized in part by the loss of high molecular weight proteins, including albumin, immunoglobulin, and transferrin, at 4 and 12 h post-infusion that resolved by 72 h. This correlated with HbG-induced GFB alterations based on the reduced expression of specific markers of podocytes (podocin, nephrin, podocalyxin, and Wilms Tumor 1 protein) and endothelial cells (ETS-related gene and claudin-5). Lectin binding studies also demonstrated marked but reversible alterations to the GFB glycocalyx accompanied by increased intraglomerular HbG deposition and 4-HNE protein adduct expression indicative of oxidative damage. Together, these findings indicate that HbG induces reversible glomerular barrier dysfunction in conjunction with transient GFB changes providing new insight into the renal response to chemically modified Hb therapeutics.

Chemical crosslinking of biopolymeric scaffolds: Current knowledge and future directions of crosslinked engineered bone scaffolds.

Bone tissue scaffolds made from either natural or synthetic polymers are employed to promote bone healing. However, lack of sufficient or poor mechanical properties such as low integrity and stability reduces their medical applications. Crosslinking, defined as induction of chemical or physical links among polymer chains, is a simple method generally used to modify mechanical, biological and degradation properties of hydrogels. Although crosslinking through chemical reactions improves the mechanical properties of bone substitutes, most of the reagents used for this aim demonstrate undesirable effects and may exert toxic reactions. Glutaraldehyde is a widely-used chemical crosslinker with unique ability to crosslink a wide variety of biomaterials; however, many contradictory views have been recently raised on its cytotoxic effects. By keeping this limit in mind, green chemicals or natural crosslinking agents have been shown to provide desired improvements in mechanical properties of bone scaffolds. Therefore, developing more efficient crosslinking materials and methods are desirable to obtain crosslinked scaffolds with perfect properties in bone tissue engineering from different biopolymers such as collagen, gelatin, cellulose, chitosan, alginate, etc. In this review, we focused on developed or developing modalities used to improve mechanical properties of various bone scaffolds and matrices based on common crosslinking reagents.

The Effect of Sterilization Methods of Endoscopic Instruments on the Body: A Study on Rat Model.

PURPOSE: Laparoscopy is widely used in many surgical areas for diagnosis and treatment. The need for sterilization of reusable instruments is an important issue. Ensuring patient safety, preventing infection, and protecting the functionality of the instruments are the most important points to be considered. We aimed to investigate two sterilization methods and their effects generated by their distribution into intra-abdominal tissues during insufflation. MATERIALS AND METHODS: 21 rats were used in the study. The Control Group (Group 1) received anesthesia for 1 hour; Group 2 (Glutaraldehyde (GA)-Pneumoperitoneum Group) received anesthesia for 1 hour; Group 3 (Ethylene Oxide (EO)-Pneumoperitoneum Group) received anesthesia for 1 hour. After 24 hours, the animals were sacrificed, and the kidneys and omentum of the animals were analyzed in a histopathological manner. Blood samples were analyzed at preoperative 24th hour and at postoperative 24th hour. RESULTS: There was a statistically significant difference in omentum, endothelium, and glomerular scores between the groups (p < 0.001 for all groups). Endothelial and glomerular scores were different at a statistically significant level in the EO and GA groups compared to the Control Group. The total score was higher at a statistically significant level in the EO and GA groups compared to the Control Group (p < 0.001 for both groups). CONCLUSION: It was determined in our study that sterilization methods such as EO and GA cause damage in intra-abdominal tissues. In the light of these results, we consider that the most ideal laparoscopic surgery set is the single-use laparoscopy set. However, this does not seem possible especially in developing countries in practice.

Evaluation of the respiratory tract toxicity of ortho-phthalaldehyde, a proposed alternative for the chemical disinfectant glutaraldehyde.

ortho-Phthalaldehyde (OPA) is a high-level chemical disinfectant that is commonly used for chemical sterilization of dental and medical instruments as an alternative to glutaraldehyde, a known skin and respiratory sensitizer. Concern for safe levels of human exposure remains due to a lack of toxicity data as well as human case reports of skin and respiratory sensitization following OPA exposure. The present study evaluated the inhalational toxicity of OPA in Harlan Sprague-Dawley rats and B6C3F1/N mice. Groups of 10 male and female rats and mice were exposed to OPA by whole-body inhalation for 3 months at concentrations of 0 (control), 0.44, 0.88, 1.75, 3.5, or 7.0 ppm. Rats and mice developed a spectrum of lesions at sites of contact throughout the respiratory tract (nose, larynx, trachea, lung), as well as in the skin and eye, consistent with a severe irritant response. In general, histologic lesions (necrosis, inflammation, regeneration, hyperplasia and metaplasia) occurred at deeper sites within the respiratory tract with increasing exposure concentration. As a first site of contact, the nose exhibited the greatest response to OPA exposure and resulted in an increased incidence, severity and variety of lesions compared to a previous study of glutaraldehyde exposure at similar exposure concentrations. This increased response in the nasal cavity, combined with extensive lesions throughout the respiratory tract, provides concern for use of OPA as a replacement for glutaraldehyde as a high-level disinfectant.

Cytotoxicity and molecular effects of biocidal disinfectants (quaternary ammonia, glutaraldehyde, poly(hexamethylene biguanide) hydrochloride PHMB) and their mixtures in vitro and in zebrafish eleuthero-embryos.

Frequently used biocidal disinfectants, including quaternary ammonium compounds (QAC), glutaraldehyde and poly(hexamethylene biguanide) hydrochloride (PHMB), occur in the aquatic environment but their potential effects in fish are poorly known, in particular when occurring as mixtures. To investigate their joint activity, we assessed the cytotoxicity of three QACs (BAC, barquat and benzalkonium chloride), glutaraldehyde andPHMB by the MTT assay individually, followed by assessing binary and ternary mixtures in zebrafish liver cells (ZFL) and human liver cells (Huh7). We also analysed molecular effects by quantitative PCR in vitro and in zebrafish eleuthero-embryos employing a targeted gene expression approach. QACs displayed strong cytotoxicity in both cell lines with EC50 values in the low µg/ml range, while glutaraldehyde and PHMB were less cytotoxic. Most of the binary and both ternary mixtures showed synergistic activity at all equi-effective concentrations. A mixture containing all five compounds mixed at their no observed effect concentrations showed strong cytotoxicity, suggesting a synergistic interaction. Additionally, we determined transcriptional alterations of target genes related to endoplasmatic reticulum (ER) stress, general stress, inflammatory action and apoptosis. Induction of ER stress genes occurred at non-cytotoxic concentrations of barquat, glutaraldehyde and BAC in ZFL cells. Barquat and BAC induced tumor necrosis factor alpha (tnf-α). Similar transcriptional alterations were found in vivo upon exposure of zebrafish eleuthero-embryos for 120h. Glutaraldehyde led to induction of ER stress genes and tnf-α, while BAC additionally induced genes indicative of apoptosis, which was also the case with benzalkonium chloride at the highest concentration. We demonstrated strong cytotoxicity of QACs, and synergistic activity of binary, ternary and quintuple mixtures. Barquat and BAC let to induction of ER stress and inflammation in vitro, and BAC and glutaraldehyde at non-toxic concentrations in vivo, while benzalkonium chloride induced expression of tnf-α only.

In vitro dentin barrier cytotoxicity testing of some dental restorative materials.

OBJECTIVES: To investigate the cytotoxicity of four dental restorative materials in three-dimensional (3D) L929 cell cultures using a dentin barrier test. METHODS: The cytotoxicities of light-cured glass ionomer cement (Vitrebond), total-etching adhesive (GLUMA Bond5), and two self-etching adhesives (GLUMA Self Etch and Single Bond Universal) were evaluated. The permeabilities of human dentin disks with thicknesses of 300, 500, and 1000µm were standardized using a hydraulic device. Test materials and controls were applied to the occlusal side of human dentin disks. The 3D-cell scaffolds were placed beneath the dentin disks. After a 24-h contact with the dentin barrier test device, cell viabilities were measured by performing MTT assays. Statistical analysis was performed using the Mann-Whitney U test. RESULTS: The mean (SD) permeabilities of the 300-µm, 500-µm, and 1000-µm dentin disks were 0.626 (0.214), 0.219 (0.0387) and 0.089 (0.028) µlmin-1cm-2cm H2O-1. Vitrebond was severely cytotoxic, reducing the cell viability to 10% (300-µm disk), 17% (500µm), and 18% (1000µm). GLUMA Bond5 reduced the cell viability to 40% (300µm), 83% (500µm), and 86% (1000µm), showing moderate cytotoxicity (300-µm) and non-cytotoxicity (500-µm and 1000-µm). Single Bond Universal and GLUMA Self Etch did not significantly reduce cell viability, regardless of the dentin thicknesses, which characterized them as non-cytotoxic. CONCLUSIONS: Cytotoxicity varied with the materials tested and the thicknesses of the dentin disks. CLINICAL SIGNIFICANCE: The tested cytotoxicity of materials applied on 300-, 500-, and 1000-µm dentin disks indicates that the clinical use of the test materials (excepting self-etching adhesives) in deep cavities poses a potential risk of damage to the pulp tissues to an extent, depending on the thickness of the remaining dentin.

Health problems have come in wake of contact with chemicals.

As a result of working with the chemicals glutaraldehyde and formaldehyde as a theatre nurse some years ago, I believe that I have suffered numerous health problems that resulted in my retirement.

Effects of Wastewater from Oil Exploration on Soil Mesofauna.

Wastewater from oil exploration may contain substances that can alter the diversity of soil organisms. This study evaluated whether produced water treated by filtration or reverse osmosis and glutaraldehyde from reverse osmosis treatments negatively affected the mesofauna in an irrigated area. In the field, irrigation with produced water treated by reverse osmosis and filtration influenced Hymenoptera and Cosmochthonius sp., while Entomobryomorpha springtails were affected only by the reverse osmosis water. In the ecotoxicological tests, reproduction in the springtail Folsomia candida was inhibited by the reverse osmosis treatment, while reproduction in the earthworm Enchytraeus crypticus was affected by both water treatments. Although glutaraldehyde did not affect the survival of F. candida, the reproduction was inhibited (EC50 = 44.4 mg/L). No adverse effect of glutaraldehyde was observed on reproduction or survival of E. crypticus. These results indicate that produced water, when used in irrigated agriculture, may affect soil functional mesofauna.

Preparation of decellularized vascular matrix by co-crosslinking of procyanidins and glutaraldehyde.

BACKGROUND: Vascular extracellular matrices (vECMs) have shown potential for small-diameter blood vessel tissue engineering applications. However, problems such as chemical instability and easy calcification are still remained. Chemical crosslinking using crosslinkers such as glutaraldehyde (GA) can improve mechanical properties and proteolysis resistance of vECMs, but leads to calcification and cytotoxicity. Procyanidins (PC) can crosslink ECMs with anti-calcification property and cytocompatibility, but the mechanical properties and chemical stability are unsatisfactory. OBJECTIVE: A novel co-crosslinking technique using PC and GA was developed, which combines the advantages of both PC and GA for enhancing mechanical properties and stability of vECMs with reduced calcification and cytotoxicity. METHODS: Fresh carotid were decellularized and then crosslinked by PC and subsequent GA for 6 h respectively. The mechanical properties, dynamic release of PC, enzymatic degradation, calcification and cytotoxicity of crosslinked samples were evaluated. RESULTS: The co-crosslinked vECMs showed enhanced tensile strength, chemical and biological stability, comparable anti-calcification property as compared to pure PC-crosslinked samples. Cytotoxicity assay showed that the co-crosslinked vECMs were cytocompatible for supporting the adhesion and proliferation of HUVECs. CONCLUSIONS: Co-crosslinking with PC and GA might be a useful method for preparation of vECM scaffolds with potential applications in small-diameter blood vessel tissue engineering.

The differential effects of heat-shocking on the viability of spores from Bacillus anthracis, Bacillus subtilis, and Clostridium sporogenes after treatment with peracetic acid- and glutaraldehyde-based disinfectants.

This study investigated (1) the susceptibility of Bacillus anthracis (Ames strain), Bacillus subtilis (ATCC 19659), and Clostridium sporogenes (ATCC 3584) spores to commercially available peracetic acid (PAA)- and glutaraldehyde (GA)-based disinfectants, (2) the effects that heat-shocking spores after treatment with these disinfectants has on spore recovery, and (3) the timing of heat-shocking after disinfectant treatment that promotes the optimal recovery of spores deposited on carriers. Suspension tests were used to obtain inactivation kinetics for the disinfectants against three spore types. The effects of heat-shocking spores after disinfectant treatment were also determined. Generalized linear mixed models were used to estimate 6-log reduction times for each spore type, disinfectant, and heat treatment combination. Reduction times were compared statistically using the delta method. Carrier tests were performed according to AOAC Official Method 966.04 and a modified version that employed immediate heat-shocking after disinfectant treatment. Carrier test results were analyzed using Fisher's exact test. PAA-based disinfectants had significantly shorter 6-log reduction times than the GA-based disinfectant. Heat-shocking B. anthracis spores after PAA treatment resulted in significantly shorter 6-log reduction times. Conversely, heat-shocking B. subtilis spores after PAA treatment resulted in significantly longer 6-log reduction times. Significant interactions were also observed between spore type, disinfectant, and heat treatment combinations. Immediately heat-shocking spore carriers after disinfectant treatment produced greater spore recovery. Sporicidal activities of disinfectants were not consistent across spore species. The effects of heat-shocking spores after disinfectant treatment were dependent on both disinfectant and spore species. Caution must be used when extrapolating sporicidal data of disinfectants from one spore species to another. Heat-shocking provides a more accurate picture of spore survival for only some disinfectant/spore combinations. Collaborative studies should be conducted to further examine a revision of AOAC Official Method 966.04 relative to heat-shocking.