Technical Services and Support Group

MICROBIAL EVALUATION of Nuron as control of Molds on Pallets from PP 300

The pallets from PP300 were subjected for Microbial evaluation due to the occuring problem of the pallet infested with molds and the need of appropriate and correct application of disinfectant. Four phased of Microbial evaluation was conducted to isolate and identify the molds in the pallet from PP300.

The four phases are:

Phase I Collection, Isolation and Identification of Molds in Pallet using Swab Method
Phase II Microbiological Assay of Fectant-3 using Serial Dilution Method
Phase III Microbiological Assay using Nuron against the Isolated and Identified Molds in pallet from P300
Phase IV Actual Preliminary Simulation of Nuron disinfectant in the PP300 Used Pallet

Phase I

Four swabs were collected from randomly selected unused and used pallets at PP 300 for Mold isolation and identification.

Table 1 – Result of Analysis of the Collected, Isolated and Identified Molds from PP300

Sample Description Molds Count (cfu/ml) Identified Molds
Fusarium sp. (cfu/ml) Aspergillus sp. (cfu/ml) Penicillum sp. (cfu/ml) Rhizopus sp. (cfu/ml)
PP 300 Unused Pallet 4.9 x 10⁵ <10 <10 <10 4.9 x 10⁵
PP 300 Used Pallet Upper Surface 2.9 x 10⁶ 1.0 x 10⁶ 1.9 x 10⁶ <10 <10
PP 300 Used Pallet Lower Surface 1.4 x 10⁶ <10 <10 1.4 x 10⁶ <10
PP 300 Used Pallet Side Surface 1.0 x 10⁵ <10 <10 <10 1.0 x 10⁵

Table 1 depicts that there were four isolated and identified Molds from the pallet swabs from PP 300. Only Rhizopus sp. tih a population count of 4.9 x 10⁵ cfu/ml was isolated from the unused pallet. On the other hand, Fusarium sp., Aspergillus sp,. Penicillum sp., and Rhizopus sp. with a population count of 1.0 x 10⁶ cfu/ml, 1.9 x 10⁶ cfu/ml, 1.4 x 10⁶ cfu/ml and 1.0 x 10⁵ cfu/ml, respectively, were isolated from the used pallet swabs.

Phase II

The four isolated and identified Molds collected from the pallets at PP300 were subjected for Microbial Assay using two disinfectant namely Fectant-3, which was the first option before apprival of Nuron, and Calsium hypochlorite using Serial Dilution Method. Thus, Fectant-3 was compared to such source of chlorine.

Fectant-3 contains 1.60% Dimethly Benezen Ammonium Chloride, 1.60% Dimethyl Ethylbenzil ammonium Chloride, 1.30% Trisodium Phosphate and 1.10% Tetrepotaasium Ethylenediamine Tetracetate labeled as sanitizer, disinfectant and deodorizer. And it is claimed to be good disinfectant for human and plant pathogen as well. Chlorine in a form of calcium hypocholrite (70%) is a common disinfectant used in our operation.

Table 2 – Result of Analysis of Microbial Assay using Serial Dilution Method

Treatment Microbial Assay using Serial Dilution Method (Density of the inoculum is 1.5 x 10⁶)
Fusarium sp. Aspergillus sp. Penicillum sp. Rhizopus sp.
Fectant-3 T1 40ml/L No growth No growth No growth No growth
T2 20ml/L No growth No growth No growth No growth
T3 10ml/L No growth No growth No growth No growth
T4 5ml/L No growth With growth With growth With growth
T5 2.5ml/L With growth With growth With growth With growth
T6 1.25ml/L With growth With growth With growth With growth
Chlorine T1 200ppm No growth No growth No growth No growth
T2 100ppm No growth No growth With growth No growth
T3 50ppm No growth No growth With growth With growth
T4 25ppm With growth With growth With growth With growth
T5 20ppm With growth With growth With growth With growth
T6 10ppm With growth With growth With growth With growth

Interpretation:

No growth – Effective
With growth – Not Effective

The Table 2 shows the different sensitivity activity of each product. Using the Fectant-3, Fusarium sp. is sensitive at 5ml/L concentration while Asperfillus sp., Penicillum sp. and Rhizopus sp. are sensitive at 10ml/L. In the case of Chlorine, Fusarium sp. is sensitive at 50ppm, Aspergillus sp. is sensitive at 25ppm, Penicillium sp. is sensitive at 200ppm and Rhizopus sp. is sensitive at 100ppm.

Therefore, Fectant-3 is effective to eliminate the four isolated molds from pallets at the rate of 10ml/L compared to chlorine at the rate of 200 ppm. Fectant-3 was originally tested and the recommend for van washing as effective control of human pathogen. However, based on the results against molds an actual simulation using Fectant-3 is to be done to evaluate the feasibility of Fectant-3 as alternative treatment for the control of molds on the pallets.

Phase III

Nuron is an organic solution of Sodium hypochlorite plus a blend of surfactants. Microbial assay was conducted using Nuron against the four molds isolated from pallets in PP300 namely: Fusarium sp., Aspergillus sp., Penicillum sp., and Rhizopus sp. using Pour Plate Method.

Table 3 – Results of Analysis of Nuron Microbial Assay using Pour Plate Method against the Isolated and identified Mold from Pallet

Treatment % EFFICACY Density of the inoculum is 1.5 x 10⁸ cfu/ml
Fusarium sp. (cfu/ml) Aspergillus sp. (cfu/ml) Penicillum sp. (cfu/ml) Rhizopus sp. (cfu/ml)
Nuron T1 100ml/900ml (10%) 99.9999 99.9997 99.9999 99.9999
T2 200ml/800ml (10%) 100 100 100 100
T3 250ml/750ml (10%) 100 100 100 100
T4 300ml/700ml (10%) 100 100 100 100
T5 400ml/600ml (10%) 100 100 100 100
T6 500ml/500ml (10%) 100 100 100 100
T7 800ml/200ml (10%) 100 100 100 100
T8 150 ppm Chlorine 99.99 99.99 99.99 99.99

Table 3 depicts that Nuron solution is ~100% effective against the four molds (Fusarium sp., Aspergillus sp., Penicillum sp., and Rhizopus sp.) with the rate ranging 20-80% solution. With the 10% Nuron solution and 150 ppm Chlorine shows 99.99% effectivity against the four molds. The preparation of the Nuron solution was in accordance to the recommended rates of the product.

Phase IV

Five used pallets from the PP300 were used for simulation trial of Nuron application. Bases on efficacy level observed in the bioassay the rates applied for simulation were 50%, 25% (based on the label recommendation) and 20% (based on the minimum inhibitory concentration) for Nuron solution compared to 150 ppm Chlorine a Ca(OCI)₂.

Simulation Procedure:

  1. Five used pallets were labelled P1, P2, P3, P4 and P5 accordingly. The received the following treatments
    P1 50% Nuron
    P2 25% Nuron
    P3 20% Nuron
    P4 150oom Chlorine
    P5 No Treatment
  2. Three 4 x 4 inch square were marked in each pallet at three specific sites : upper, lower and side surface which were as sample sites for the swab collection.
  3. A spore suspension was prepared from each of the four molds collected from the pallet for inoculation.
  4. 500 ml of each 50%, 25% and 20% Nuron and 500ml of chlorine solution was prepared and placed into a Solo sprayer.
  5. Swab method was used for the simulation trial. Each of the marked squares was swabbed prior to inoculation with mold inculum.
  6. About 7.5 ml of the spore suspension was inoculated in each marked squares in each pallet.
  7. After two hours of inoculation each of the marked squares were swabbed.
  8. Application of each treatment was done according to testing protocol.
  9. 5 minutes after treatment application the marked squares were swabbed.
  10. Consecutive swabbing was done after 24 hours, 48 hours and 72 hours.
  11. All collected swabs were run for mold count.

SUMMARY:

The results depict that both unused and used pallets from PP300 were infested with molds namely Fusarium sp., Aspergillus sp., Penicillum sp., and Rhizopus sp. Microbiology assay was conducted to determine the preferable disinfectant to be used for washing the infested pallets. Among the tested concentration 10ml/L of Fectant-3, 200ppm of Chlorine and 20% (200ml/800ml) concentration of Nuron were effective in eliminating the four identified molds from pallets in PP300.

The results of the Mold count (cfu/ml) in the actual preliminary simulation using the pallets from PP300 depict that Nuron as disinfectant was ~100% effective in eliminating the molds at the rate of 50%, 25% and 20%. The efficacy of Calcium hypochlorite in a 150 ppm Chlorine solution was ~84% effective.

RECOMMENDATION:

The product Nuron with a concentration of 20-50 % showed efficacy to control molds on pallets. It is comparable to the efficacy of Fectant-3 at 10ml/L and 200ppm of Calcium Hypochlorite. It control the four identitified molds found on the pallets from PP300.

The recommendation by the manufacturer for decks and pallets is to begin at the high point. They say to work in sections and make sure all surfaces are covered. Then the mold and mildew, algae/moss remover will begin to work instantly and as the water recedes it will pulled down through the surface. The recommend to let the area soak for 2 mins up to 10 mins, no need to rinse. They also indicate to remove fungus from wood pallets and prevents mold and mildew in new pallets.

The also recommend that “for best results on a porous surface, hose down first and wait until all surface water drains off leaving a damp look. The product can be applied with a hand sprayer, watering can. Or simply poured on and moved around with a broom. Heavily affected areas may need a second application or a rub with a stiff bristle broom to loosen the build up.

Since under our conditions the hand sprayer will result impractical we recommend the piling of the pallets and the usage of a car was pressurized system of spraying with the concentration of 25% Nuron solution (based on actual CFU counts) to cover even the human pathogen that could be present.

During the actual simulation of Nuron solution it was observed that the solution has a strong odor, therefore it is a must that the cleaning/washing area for pallets should be well ventilated and the worker should strictly wear proper and correct PPE (coverall suite similar as the WTS Packing house PPE) using particularly N95 mist respiratory mask, a NIOSH approved type of face mask.

Prepared by:

Florence-B-Alzate

 

 

 

Florence B Alzate
MMBL Head

Noted and Approved by:

Dr.-Luis-H.-Jacome

 

 

 

 

Dr. Luis H. Jacome
TSSG Director

Table 4 – Mold Count (cfu/ml) Results of the Actual Preliminary Simulation of PP300 used Pallet

Pallet No. Treatment Swab Description Swab Area Results (Mold count cfu/ml)
P1 50% Nuron Before inoculation of Mold Upper 71000
Lower 6300
Side 5300
After 2 hours of inoculation Upper 1400000
Lower 1400000
Side 1200000
After 5 mins. application of Treatment Upper 100
Lower <10
Side <10
After Day 1 Upper 2200
Lower <10
Side 1000
After Day 2 Upper 2200
Lower 450
Side 850
After Day 3 Upper 800
Lower 450
Side 2800
P2 25% Nuron Before inoculation of Mold Upper 1500
Lower 2400
Side 2000
After 2 hours of inoculation Upper 90000
Lower 80000
Side 100000
After 5 mins. application of Treatment Upper <10
Lower 700
Side <10
After Day 1 Upper 400
Lower 1300
Side 200
After Day 2 Upper 2100
Lower 1200
Side 600
After Day 3 Upper 2800
Lower 750
Side 1700
P3 20% Nuron Before inoculation of Mold Upper 12000
Lower 130000
Side 16000
After 2 hours of inoculation Upper 490000
Lower 460000
Side 510000
After 5 mins. application of Treatment Upper <10
Lower 900
Side 800
After Day 1 Upper 4900
Lower 3300
Side 11000
After Day 2 Upper 110000
Lower 11000
Side 14000
After Day 3 Upper 100
Lower 25000
Side 4600
P4 150 ppm Chlorine Before inoculation of Mold Upper 20000
Lower 110000
Side 1100000
After 2 hours of inoculation Upper 1100000
Lower 1000000
Side 1400000
After 5 mins. application of Treatment Upper 250000
Lower 40000
Side 300000
After Day 1 Upper 410000
Lower 230000
Side 530000
After Day 2 Upper 440000
Lower 470000
Side 560000
After Day 3 Upper 450000
Lower 530000
Side 630000
P5 No treatment Before incubation Upper 2200
Lower 25000
Side 6100
After Day 1 Upper 2800
Lower 28000
Side 8200
After Day 2 Upper 6100
Lower 53000
Side 15000
After Day 3 Upper 7500
Lower 60000
Side 28000
nuronFusarium Oxysporum Report