Technical Services and Support Group

November 14, 2012

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
MMBL Head

Noted and Approved by:

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

A. Growth of FoC @ diff. level of NB conc.