Cellulose Nanocrystal 

Entrapment of Benzalkonium 

Chloride in Southern Pine

Biological, Chemical, and Physical 

Properties

Carol A. Clausen

Vina W. Yang 

Rachel A. Arango

Laura E. Hasburgh

Patricia K. Lebow

Richard S. Reiner

Research Paper

FPL–RP–680

United States Department of Agriculture

Forest 

Service

Forest Products

Laboratory

September

2015

CNC/ADBAC ADBAC/CNC
September 2015

Clausen, Carol A.; Yang, Vina W.; Arango, Rachel A.; Hasburgh, Laura E.; 

Lebow, Patricia K.; Reiner, Richard S. 2015. Cellulose nanocrystal entrapment of benzalkonium chloride in southern pine: Biological, chemical, 

and physical properties. Research Paper FPL-RP-680. Madison, WI: U.S. 

Department of Agriculture, Forest Service, Forest Products Laboratory. 8 p.

A limited number of free copies of this publication are available to the 

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Abstract

Protecting wood products from biodeterioration has 

been a dynamic area of research in the past decade with 

an emphasis on the development of non-arsenical wood 

preservatives. Naturally occurring cellulose nanomaterials 

that are reported to have unique chemical properties, high 

strength, and stiffness were evaluated for the potential 

to improve durability of wood either as a stand-alone 

treatment or in combination with a cationic biocide. In this 

study, benzalkonium chloride (ADBAC) was selected as 

a biocidal counter-cation for the sulfate ester on cellulose 

nanocrystals (CNC). In a two-step pressure treatment, 

specimens of southern pine were impregnated with CNC 

or ADBAC to form a CNC/ADBAC hybrid agglomerate 

within the wood. Cellulose nanocrystals penetrated wood 

in the CNC control and when CNC was the first step in a 

dual treatment with ADBAC. However, when ADBAC was 

the first step in a dual treatment with CNC or pre-mixed 

with CNC, the agglomerated ADBAC/CNC could not 

penetrate wood and gelled on the wood surface. Biological 

and chemical analysis of wood treated with the CNC/

ADBAC hybrid revealed that CNC provided no additional 

resistance to fungal colonization, termite attack, or chemical 

leaching when agglomerated to ADBAC in wood. Analysis 

of modulus of elasticity, modulus of rupture, work to 

maximum load, and mass loss during combustion showed 

no change in the physical properties from the addition of 

CNC to wood impregnated with the CNC/ADBAC hybrid 

Contents

Introduction......................................................................1

Materials and Methods.....................................................1

Chemicals .....................................................................1

Fungal Inoculum...........................................................1

Agar Toxicity Test.........................................................2

CNC/Biocide Hybrid Characterization Metrics............2

Wood Treatment............................................................2

Biological Evaluation of CNC/ADBAC 

Hybrid Treatment..........................................................3

Chemical Retention ......................................................3

Physical Properties........................................................3

Results and Discussion ....................................................4

Agar Toxicity Screening ...............................................4

Characterization Metrics...............................................4

Treatment Retention .....................................................5

Leach Resistance ..........................................................5

Biological Resistance....................................................6

Physical Properties........................................................7

Conclusions......................................................................7

Acknowledgments............................................................8

References........................................................................8

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treatment. While results of this study did show penetration 

of CNC into southern pine and leach-resistant binding 

of CNC to ADBAC, neither treatment of southern pine 

with CNC nor the addition of CNC to ADBAC in a dual 

treatment improved the biological, chemical, or physical 

durability properties of the wood.

Keywords: Cellulose nanocrystals (CNC), brown-rot fungi, 

mold fungi, subterranean termites, wood preservation, 

nanoentrapment, benzalkonium chloride (ADBAC), MOR, 

MOE, mass loss calorimetry
Cellulose Nanocrystal Entrapment of 

Benzalkonium Chloride in Southern Pine

Biological, Chemical, and Physical Properties

Carol A. Clausen, Supervisory Research Microbiologist

Vina W. Yang, Microbiologist

Rachel A. Arango, Entomologist

Laura E. Hasburgh, Fire Protection Engineer

Patricia K. Lebow, Mathematical Statistician

Richard S. Reiner, Chemical Engineer

Forest Products Laboratory, Madison, Wisconsin

Introduction

Leach resistance is a critical factor in development of new 

wood preservative formulations. Wood preservatives that 

generally resist leaching during service rely on chemical 

fixation reactions based on drying time and temperature that 

take place after the wood is impregnated with the chemical 

following pressure treatment (Lebow 1996). Wood preservatives that are readily leachable, like borates, have limited 

applications. Improving the leach resistance of soluble biocides has remained an area of interest to the wood preservation industry prompting the need for novel approaches to 

wood treatment with leachable preservative chemicals.

Nanomaterials exhibit unique physical and physiochemical properties that differ significantly from larger particles 

of the same material (Clausen 2007; Clausen et al. 2012). 

Cellulose nanocrystals (CNC) are reported to demonstrate 

very high strength and stiffness properties that can impart 

improved strength to composites and coatings (Wegner 

et al. 2005; Rains et al. 2014). High-yield cellulose nanocrystal production has recently been achieved on a scalable 

level through optimization of acid hydrolysis (Wang et al. 

2012). Two things happen during acid hydrolysis: First, the 

amorphous region of cellulose is largely hydrolyzed, but the 

crystalline regions remain intact, and second, the conditions 

are such that some of the primary alcohols at the CNC surface are converted to negatively charged sulfate esters. The 

resulting CNCs are uniformly dispersible; the dispersion 

stability of CNCs coupled with the controlled uniformity 

results in a nanomaterial that should be able to penetrate 

wood in an aqueous dispersion. The negatively charged sulfate ester groups are suitable for ionic binding with cationic 

quaternary ammonium compounds. 

Quaternary and tertiary amine salts, collectively known as 

alkylammonium compounds (AAC), have long been recognized as effective biocides against most decay fungi and 

Reticulitermes sp. (Preston and Nicholas 1982; Preston et al. 

1987) with didecyldimethyl ammonium chloride (DDAC) 

most commonly used in wood preservation formulations 

during the 1990s. In the early 2000s, benzalkonium chloride (ADBAC) was added as a co-biocide to copper monoethanolamine to create one of several alkaline copper quat 

(ACQ) wood preservative formulations (Lebow 2004). We 

hypothesize that soluble quaternary ammonium compounds 

bound to CNC within wood may serve to simultaneously 

prevent leaching and provide improved physical properties 

to the wood.

This study had four objectives: 1) screen and select candidate quaternary compounds for optimal binding and efficacy, 2) evaluate efficacy of a CNC/quat hybrid against 

biological organisms that attack wood in-service, 3) assess 

penetration of wood and resistance to leaching of CNC/quat 

hybrid, and 4) determine whether a CNC/quat hybrid alters 

physical properties of wood following impregnation with 

the CNC/biocide hybrid molecule.

Materials and Methods

Chemicals

A range of concentrations of four cationic surfactants were 

screened for antifungal activity against mold and decay test 

fungi: benzalkonium chloride (ADBAC), cetrimonium bromide (CTAB), and octadecyltrimethylammonium bromide 

(OTAB) from Sigma-Aldrich, Inc., St. Louis, Missouri, and 

benzethonium chloride (BZT), Lonza, Inc., Allendale, New 

Jersey. 

Fungal Inoculum

Mold fungi

Three mold fungi, Aspergillus niger Tiegh. (2.242) provided 

by University of Virginia, Penicillium chrysogenum Thom. 

(PH02) from the Center for Forest Mycology Research, 

Wisconsin, and Trichoderma atraviride Bissette (ATCC 

20476), were grown on 2% malt extract agar (MEA) (Difco, 

Becton, Dickinson & Company, Maryland) for 2 weeks. 

Spore suspensions of test fungi were prepared by washing the surface of each malt agar plate with 10–15 mL of 

sterile deionized water (DI) according to ASTM standard 
Research Paper FPL–RP–680

2

D4445-10 (ASTM 2011). Individual mold spore suspensions 

were transferred to a spray bottle and diluted to 100 mL 

with DI water to yield approximately 3 × 107 spores/mL. 

The spray bottles were adjusted to deliver 1-mL inoculum 

per spray. 

Decay fungi

Brown-rot fungi, Gloeophyllum trabeum (Pers.: Fr.) Murr 

(MAD 617) and Postia placenta (Fr.) M. Lars et Lomb 

(MAD 698), or Fibroporia radiculosa (Peck) Gilb. & 

Ryvarden (TFFH 294) and a white-rot fungus, Trametes 

versicolor (L. Fr.) Pil. (MAD 697) were used to screen 

chemical and for resistance testing. Test fungi obtained from 

Forest Products Laboratory, Wisconsin, were maintained on 

2% MEA. Agar plugs were taken from the edge of active 

mycelial growth from individual fungal cultures and separately blended with 10 mL sterile DI water to create a liquid 

fungal inoculum. 

Agar Toxicity Test

One mL of fungal inoculum was sprayed (for mold fungi) or 

spread (for decay fungi) over the surface of three MEA Petri 

dishes. Aqueous dilutions of 0.5, 1.0, 2.0, 5.0, and 10% 

(w/vol) active ingredient were prepared for each cationic 

surfactant. Filter paper discs (13 mm diameter) were saturated with a 10-μl aliquot for individual concentrations of 

test chemicals and placed in the center of the pre-inoculated 

Petri dishes. Petri dishes were incubated at 27 °C and 70% 

relative humidity (RH) for 2 weeks or until the control 

plates were substantially covered with fungal growth. Clearing zones surrounding the chemical-impregnated discs were 

measured as the zone of inhibition (ZOI) and recorded after 

subtracting the diameter of the filter paper disc. Based on 

the results of the agar toxicity test, the most efficacious 

surfactant (referred to as ADBAC from here forward) was 

selected for the subsequent experiments. 

CNC/Biocide Hybrid Characterization Metrics

Cellulose nanocrystals (CNC) were produced at the Forest 

Products Laboratory, Wisconsin (Wang et al. 2012). The optimal ratio of biocide to CNC was determined through observation of agglomeration and weight of the CNC/ADBAC 

hybrid (Table 1). Visual observations included the amount 

of settling that occurred upon mixing various ratios of CNC 

and the biocide, ease of resuspending the settled hybrid and 

clarity or cloudiness of the sample. To test that biocide efficacy was retained following binding to CNC, equal volumes 

of CNC were gently mixed with various concentrations of 

ADBAC and held at room temperature for 24 h. Ten microliters of the resulting CNC/ADBAC hybrid were placed at 

the center of agar plates that were freshly inoculated with 

fungal inoculum. Plates were incubated at 27 °C, 70% RH 

for 3 weeks. ZOI diameters surrounding the CNC/biocide 

hybrid were recorded when present. Wood treated with the 

CNC/biocide hybrid was evaluated by energy dispersive 

x-ray analysis (EDAX), inductively coupled plasma (ICP) 

and scanning electron microscopy (SEM) for the presence 

of biocide and CNC. 

Temperature stability

Aliquots of the CNC/ADBAC hybrid mixture were placed 

in four separate tubes and stored at 0, 10, 25, and 50 °C for 

24 h. Ten microliters of each aliquot were evaluated for agar 

toxicity. 

pH stability

Aliquots of the CNC/ADBAC hybrid were adjusted with 1N 

HCl and 1 M NaOH to pH 3.0, 7.0, and 9.0. Ten microliters

of each pH-adjusted aliquot were evaluated for agar toxicity 

as described above. 

Wood Treatment

Vacuum impregnation

Southern pine sapwood, 7 × 20 mm cross section × 7 cm 

long for mold tests, 1 × 1 × 1 cm for decay tests, 25 × 25 × 

5 mm for termite tests and 100 mm × 100 mm for mass loss 

calorimetry were vacuum impregnated in a series of sequential two-step treatments described in Table 2. Each treatment consisted of exposing specimens to the first chemical 

under vacuum for 20 min at 172 kPa followed by exposing 

the same specimens to the second chemical under vacuum 

for an additional 20 min at 172 kPa. Specimens were blotted dry on paper towels between treatments. Controls were 

subjected to dual treatments with DI water. Following dual 

Table 1—Binding, stability, and characterization metrics 

for CNC/biocide hybrid

Biocide 

concentration

(%)

CNC/

biocide

ratio

Temperature

stability

(°C)

Binding 

stability

(pH)

Visual 

observations

Characterization

metrics

0.1 1:1 0 3.0 Settling Dehydration/

rehydration

0.5 1:2 10 7.0 Resuspension EDAXa

1.0 2:1 25 9.0 Optics ICPb

2.0 50 SEMc

a

Energy Dispersive X-ray Analysis. b

Inductively coupled plasma. c

Scanning electron microscopy.
Cellulose Nanocrystal Entrapment of Benzalkonium Chloride in Southern Pine: Biological, Chemical, and Physical Properties

3

treatment, specimens were air-dried for 72 h prior to conditioning at 27 °C, 70% RH for 2 weeks.

Vacuum-pressure impregnation

Southern pine sapwood stakes (1.9 × 1.9 × 40.6 cm) were 

submerged in treatment solution 1 (Table 2) for 40 min 

vacuum followed by 345 kPa for 40 min. Each stake was 

blotted dry, weighed and submerged in treatment solution 2 

(Table 2) for 40 min vacuum followed by 345 kPa for 

40 min. Controls were subjected to dual water treatments 

under the same vacuum and pressure regime. Following 

dual treatment, specimens were air-dried for 2 weeks and 

conditioned at 21 °C and 70% RH for 4 weeks prior to 

testing.

Biological Evaluation of CNC/ADBAC 

Hybrid Treatment

Mold resistance

Six randomly selected southern pine specimens per treatment group were sprayed with 1-mL spore inoculum and 

incubated at 27 °C and 80% RH for 4 weeks. Untreated 

southern pine served as the control. Following incubation, 

specimens were visually rated on a scale of 0–5 with 0 

indicating the specimen was completely free of mold growth 

and 5 indicating the specimen was completely covered with 

mold growth.

Decay resistance

Following the guidelines of AWPA standard E10‒12 (AWPA 

2014), five treated and five untreated southern pine specimens per treatment group were weighed and placed in a 

soil-block test. Trametes versicolor was replaced with 

Fibroporia radiculosa (Peck) Gilb. & Ryvarden (TFFH 294)

to test decay resistance of the CNC/ADBAC hybrid in wood 

blocks due to the unique preservative tolerance properties 

of F. radiculosa of importance to the wood protection industry (Clausen and Jenkins 2011). Soil bottles were prepared 

by inoculating a pine feeder with one of three brown-rot 

test fungi, G. trabeum, P. placenta, and F. radiculosa, and 

incubated at 27 °C and 80% RH for 12 weeks. Following incubation, fungal mycelium was brushed from the specimens, 

specimens were oven-dried, reconditioned at 27 °C and 80% 

RH, and reweighed. Percentage mass loss was calculated for 

each specimen and reported as an average for each treatment 

group.

Termite resistance

A no-choice termite test with Reticulitermes flavipes (Kollar) (Eastern subterranean termites) was performed using 

five unleached specimens per treatment and five untreated 

controls that were pre-weighed and conditioned at 27 °C 

and 70% RH. Termites were collected from Janesville, Wisconsin. One specimen was placed in the bottom of an acrylic 

cylindrical container (90 mm diameter and 60 mm tall) with 

1 g of R. flavipes and moist sand. The containers were maintained at 27 °C and 85% RH for 4 weeks based on AWPA 

E1‒13 standard method (AWPA 2014). Tests were periodically checked for moisture and mortality. At the end of the 

incubation, wood specimens were oven-dried, reconditioned 

at 27 °C and 70% RH, and reweighed to calculate mass loss.

The termite mortality rate was estimated by counting the 

remaining live termites. Following incubation, wood specimens were visually rated on a scale of 0–10 with 10 being 

sound and 0 equaling failure. 

Chemical Retention

Analytical assessment

Southern pine specimens that were treated with CNC, 

ADBAC, or combinations of CNC and ADBAC were assessed with scanning electron microscopy (SEM), EDAX 

analysis, and inductively coupled plasma (ICP) for treatment penetration and retention following leaching.

Leaching resistance

Leaching procedures were similar to AWPA E11‒12 standard method (AWPA 2014). Five specimens per treatment 

were placed into individual 500-mL beakers, submerged in 

100 mL of DI water and subjected to mild agitation for a 

total of 14 days, with a complete change of water after 6 h, 

and 1, 2, 4, 6, 8, 10, 12, and 14 days. Leached specimens 

were dried, reconditioned for 2 weeks at 27 °C and 80% 

RH, and evaluated for mold and decay resistance. Leached 

specimens were also subjected to ICP analysis.

Physical Properties

Mechanical properties

Modulus of elasticity (MOE) and modulus of rupture 

(MOR) were assessed, and work to maximum load (WML) 

was calculated on 30 specimens for each treatment group 

according to ASTM D143‒14 (ASTM 2014). Work to maximum load is a measure of the combined strength and toughness of the wood under bending stress. Specimens were conditioned at 21 °C and 70% RH for 4 weeks prior to testing. 

Based on simple fixed effect models, the mean MOE, MOR, 

and WML for each group of test specimens were compared 

Table 2—Chemical retention in southern pine 

double treated with CNC or ADBAC

Treatment 1 Treatment 2

Average retention

(kg/m3)

DI H2O DI H2O 0

CNC CNC 5.52

ADBAC ADBAC 18.20

CNC ADBAC 15.71

ADBAC CNC 1.98

Pre-mixed 

CNC/ADBAC

Pre-mixed 

CNC/ADBAC

2.57
Research Paper FPL–RP–680

4

with the mean values of control specimens to determine 

statistical significance of differences following treatment 

of wood with CNC, ADBAC, or combinations of CNC and 

ADBAC using SAS® 9.4. 

Combustion characteristics

Specimens for each treatment group were tested in triplicate 

using the mass loss calorimeter (MLC). The MLC is commonly used as the first step in obtaining combustion and 

flammability characteristics of materials such as the heat 

release rates (HRR), effective heat of combustion, and mass 

loss rates. The MLC uses a truncated conical electric heater 

that provides a constant heat flux onto a test specimen, and 

after piloted ignition by the spark igniter, the mass loss and 

HRR are recorded. The HRR is determined by digitally deconvolving the signal of the chimney wall thermopile and 

combining the resulting processed signal with the standard 

gas convection thermopile signal into a composite value 

(Dietenberger and Boardman 2013). The 100- × 100-mm 

test specimens were subjected to irradiance at 50 kW/m2 in 

a horizontal orientation. Prior to testing, the samples were 

conditioned at 21 °C and 50% RH for 4 weeks. The data 

were analyzed and the averages of the triplicates were compared with the average values of the water-treated control 

specimens. 

Results and Discussion

Agar Toxicity Screening

Agar toxicity test results are summarized in Figures 1 

and 2. Of the four cationic quaternary ammonium compounds screened for anti-fungal properties, ADBAC at a 

concentration > 2% had the greatest ZOI for the mold fungi 

(Fig. 1) selected for this study; ADBAC showed the greatest 

ZOI at all concentrations for the decay fungi (Fig. 2).

Characterization Metrics

Binding characteristics

Visual examination of flocculation patterns for CNC and 

ADBAC at ratios of 2:1, 1:1, 1:5, and 1:10 after 24 h at 

25 °C showed that a 1:1 ratio produced a semi-solid pellet 

without any unbound liquid (Table 1). At higher ratios of 

CNC, less than optimal binding occurred resulting in a liquid dispersion with a high flow rate. Lower ratios of CNC 

to ADBAC resulted in a solid pellet with notable amounts 

of unbound liquid. The 1:1 ratio was selected for further 

experiments. Theoretically, CNC/ADBAC agglomeration should facilitate physical entrapment of the otherwise 

soluble biocide within the wood but without modifying the 

wood matrix. 

Dispersibility is generally essential for CNC storage and 

transport. Beck et al. (2012) reported that above a threshold 

moisture content of 4%, dried CNC is fully dispersible in 

water. The CNC/ADBAC hybrid readily rehydrated after 

dehydration. Dried preparations of CNC/ADBAC were dispersible, but not necessarily dissociated in water. 

Stability tests

For stability tests, CNC/ADBAC was adjusted to pH 3.3, 

7.0, or 9.0 before testing for ZOI in a mold inhibition test. 

Results demonstrated similar ZOIs for mold test fungi at 

each pH; therefore, pH 7.0 was selected for further experiments. The initial pH of CNC was 7.38. 

0.0

0.5

1.0

1.5

2.0

2.5

Zone of inhibition (cm)

0.5%

1%

2%

5%

10%

BZT CTAB OTAB

T. atraviride

BZT CTAB OTAB ADBAC

A. niger

ADBAC BZT CTAB OTAB

P. chrysogenum

ADBAC

Figure 1—Inhibition of three mold fungi by benzalkonium chloride (ADBAC), benzethonium chloride (BZT), 

cetrimonium bromide (CTAB), and octadecyltrimethylammonium bromide (OTAB) in an agar toxicity test.
Cellulose Nanocrystal Entrapment of Benzalkonium Chloride in Southern Pine: Biological, Chemical, and Physical Properties

5

0

1

2

3

5

6

BZT CTAB OTAB

4

Zone of inhibition (cm)

0.5%

1%

2%

5%

10%

ADBAC ADBAC BZT CTAB OTAB ADBAC BZT CTAB OTAB

G. trabeum P. placenta T. versicolor

Figure 2—Inhibition of two brown-rot fungi (G. trabeum and P. placenta) and one white-rot fungus (T. versicolor) 

by benzalkonium chloride (ADBAC), benzethonium chloride (BZT), cetrimonium bromide (CTAB) and 

octadecyltrimethylammonium bromide (OTAB) in an agar toxicity test.

CNC/ADBAC was also exposed to temperatures ranging 

from 0 °C to 50 °C for 24 h before testing for ZOI in a mold 

inhibition test. Results showed that ZOIs were the same or 

slightly higher at 25 °C; therefore, 25 °C was selected for 

further experiments. 

Treatment Retention

The dual treatment process consisted of two subsequent 

treatments in separate chemical solutions (Table 2). The percentage of chemical uptake was calculated. CNC penetrated 

southern pine resulting in a 5.52 kg/m3 uptake (n = 5). Dual 

treatment with ADBAC and dual treatment with CNC followed by ADBAC resulted in 18.2 and 15.7 kg/m3

 uptake, 

respectively. When wood was treated with ADBAC followed by CNC or when the ADBAC and CNC were premixed before pressure impregnation, the treatment visibly 

agglomerated on the surface of the wood specimens and 

prevented appreciable penetration (Fig. 3).

Figure 3—Comparison of penetration for CNC/ADBAC 

(left) and ADBAC/CNC (right) in southern pine.

EDAX analysis of treated wood specimens was not sensitive enough to estimate treatment chemical uptake utilizing 

chlorine as the marker for ADBAC and sulphur as markers 

for CNC. 

Leach Resistance

Binding visibly occurred upon physical contact of CNC 

with ADBAC to form an agglomeration that appeared to 

coat the bordered pits of southern pine (Fig. 4). Leached and 

unleached southern pine specimens treated with CNC and 

ADBAC were ground to 20 mesh (nominal 841 μm) and 

analyzed using inductively coupled plasma (ICP) spectroscopy for chlorine and sulphur. The liquid solution of CNC 

and ADBAC used for treatment was also analyzed. Results 

showed that approximately 9% of CNC in solution penetrated into wood with 66.7% of the CNC in wood remained 

after leaching (Table 3). For the ADBAC, 12.6% of the 

treating solution penetrated the wood with 48% remaining 

after leaching. Chlorine levels were not detectable in the 

Figure 4—Scanning electron micrograph of southern pine 

bordered pits (arrow) impregnated with CNC/ADBAC hybrid.
Research Paper FPL–RP–680

6

CNC/ADBAC mixture in the wood, but 18% of the CNC 

measured as sulphur was detected in both unleached and 

leached samples, suggesting the CNC bound to ADBAC 

within the wood and did not leach out.

Although ADBAC is water soluble, Lee and Cooper (2010) 

reported two adsorption mechanisms for ADBAC onto 

wood: ion exchange at low concentration and hydrophobic 

interaction at high concentrations. They reported that when 

used without copper, the adsorption capacity of ADBAC 

through ion exchange does not achieve the Na+ cation exchange capacity of wood. The leaching results in this study 

suggest penetration of the wood by CNC and binding within 

the wood of CNC/ADBAC. The ADBAC served as the 

counter cation for the sulfate ester on the CNC and is ionically bound to the CNC, so leach resistance is essentially 

a measure of the dissociation constant of the CNC-sulfateADBAC salt. 

Biological Resistance 

Treated leached southern pine specimens were tested for 

resistance to biological attack by mold fungi (Fig. 5), decay 

fungi (Fig. 6), and subterranean termites (Fig. 7). Results of 

the mold and decay tests revealed no change in the biological resistance of southern pine when CNC was present in 

the chemical treatment compared to the chemical treatment 

alone. All treatments that included ADBAC showed similar 

levels of fungal inhibition regardless of the treatment group. 

Results for the termite no-choice test showed a 20% weight 

loss in water controls, 25% weight loss in CNC controls, 

and only 2% to 4% weight loss in all samples treated with 

ADBAC regardless of the treatment group. There was little 

mortality in the termite test and all specimens that were 

treated with ADBAC showed visible repellency to termite 

tunneling. 

Table 3—ICP chlorine and sulphur analysis of 

unleached and leached southern pine treated with 

CNC and ADBAC alone and in combination

Unleached

(ppm)

Leached

(ppm)

Cl S Cl S

Treating solution

CNC NDa 3,300 – –

ADBAC 60,900 ND – –

CNC/ADBAC 28,700 1,650 – –

Treated pine

CNC ND 300 ND 200

ADBAC 7,700 50 3,700 30

CNC/ADBAC ND 300 ND 300

Untreated control ND 40 – –

a

ND, none detected.

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

Mold rating

DI water CNC ADBAC CNC/

ADBAC

4 wk

8 wk

12 wk

Figure 5—Mold growth after 4-, 8-, and 12-week exposure of 

leached CNC/ADBAC-treated southern pine to a mixed mold 

spore suspension. 

70

60

50

40

30

20

10

0

Weight loss (%)

DI 

water

CNC CNC/

ADBAC

ADBAC/

CNC

ADBAC

P. placenta

G. trabeum

F. radiculosa

Figure 6—Weight loss in leached CNC/ADBAC-treated 

southern pine following exposure to Postia placenta, 

Gloeophyllum trabeum, and Fibroporia radiculosa.

30

25

20

15

10

5

0

Weight loss (%)

DI 

water

CNC CNC/

ADBAC

ADBAC/

CNC

premix

CNC/

BAC 

premix

ADBAC

Figure 7—Weight loss in leached CNC/ADBAC-treated 

southern pine in a no-choice test against Reticulitermes 

flavipes subterranean termites. 
Cellulose Nanocrystal Entrapment of Benzalkonium Chloride in Southern Pine: Biological, Chemical, and Physical Properties

7

Physical Properties

Mechanical testing

Mean MOR, MOE, and WML and their associated group 

estimated standard errors are summarized in Table 4. Group 

differences were not detected for MOE (p-value = 0.2201), 

MOR (p-value = 0.1727), and WML (p-value = 0.1537). 

Results did not reveal any statistical differences between 

the treatment groups suggesting that penetration of southern pine with CNC under the conditions of this study does 

not significantly increase or decrease strength, stiffness, or 

toughness. For each property, a one-way analysis of variance was conducted using SAS® 9.4. Dunnett’s test was 

used to adjust p-values for pairwise comparisons of each 

treatment group with the controls. Results of the comparison 

showed no statistical differences between the controls and 

any of the treatment groups (p < 0.05). Agglomerations of 

CNC in a dispersed solution are reported to weaken the mechanical properties of the CNC (Steele et al. 2012), but it is 

not known whether agglomeration because of ionic bonding 

within a wood matrix would demonstrate similar mechanical 

properties. 

Combustion properties

Mass loss calorimetry results showed no appreciable difference between controls and treated specimens regarding effective heat of combustion, mass loss rate, and heat release 

rates for southern pine pressure-treated with CNC and 

ADBAC alone and in combination (Fig. 8). 

Conclusions

Conditions used to produce cellulose nanocrystals (CNC) 

result in formation of sulfate ester groups that are suitable 

for ionic binding with soluble cationic quaternary ammonium compounds. CNC were evaluated for their potential 

to bind and entrap benzalkonium chloride (ADBAC), a 

quaternary ammonium compound, in southern pine. Penetration and entrapment of CNC and ADBAC in wood were 

Table 4—Mechanical properties of unleached southern pine pressure treated 

with CNC or ADBAC

Treatment 1 Treatment 2

MOE

(MPa)

MOR

(kPa)

WML

(J)

Mean S.E.a Mean S.E. Mean S.E.

DI H2O DI H2O 16,500 363.8 112,054 2,298 12.78 0.553

CNC CNC 16,174 363.8 118,156 2,298 12.54 0.553

ADBAC ADBAC 17,180 370.0 114,474 2,337 13.06 0.563

CNC ADBAC 16,450 363.8 114,577 2,298 14.12 0.553

ADBAC CNC 15,907 363.8 119,079 2,298 12.01 0.553

Pre-mixed

CNC/ADBAC

Pre-mixed

CNC/ADBAC

16,684 363.8 118,783 2,298 12.64 0.553

a

S.E., Standard Error; note one observation from the ADBAC/ADBAC group experienced a brash tension

failure and was excluded.

Figure 8—Average heat release rate (HRR) in kW/m2 from 

triplicates of each treatment obtained by the modified mass 

loss calorimeter. 

demonstrated under the conditions of this study. Treated 

wood specimens were assessed for changes in biological, 

chemical, and physical properties. It was shown that 

ADBAC alone provided sufficient efficacy against mold 

fungi, decay fungi, and subterranean termites in leached 

specimens. No statistical differences were detected in 

strength (MOR), stiffness (MOE), or toughness as measured 

by work to maximum load between control specimens and 

those that were treated with CNC or ADBAC alone or in 

combination. Likewise, specimens treated with CNC or 

ADBAC alone or in combination did not demonstrate 

changes in combustion as measured by mass loss and heat 

release rate compared to control specimens. In conclusion, 

southern pine treated with cellulose nanocrystals agglomerated to benzalkonium chloride (ADBAC) within the wood, 

but the CNC/ADBAC hybrid molecule does not appreciably 

alter biological durability, nor does it statistically improve 

mechanical properties, or combustion resistance compared 

to untreated controls.

0

20

40

60

80

100

120

140

0 50 100 150 200 250 300 350

HRR (kW/m2

)

Time (s)

ADBAC/ADBAC

CNC/CNC

ADBAC/CNC

CNC/ADBAC

PRE/PRE

H2O/H2O
Research Paper FPL–RP–680

8

Acknowledgments

Authors thank Steve Halverson, Physical Science Technician, for wood treatments; Tim Nelson, Mechanical Engineering Technician, and Marshal Begel, General Engineer, 

for conducting mechanical testing and analysis; Kolby 

Hirth, Chemist, for ICP analysis; and Tom Kuster, Forest Products Technologist, for preparing scanning electron 

micrographs.

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