Environmental and Toxicological Analysis
Report for PERMA-ZYME
TM

December 9, 2002
Prepared by:

Civil Engineering Research Foundation/International Institute of
Energy Conservation (CERF/IIEC)
2131 K Street NW, Suite 700
Washington, DC 20037-1810
202-785-6420

1. Introduction

The Civil Engineering Research Foundation/International Institute of Energy
Conservation (CERF/IIEC) has signed a contact with Federal Highway Administration to evaluate the performance of soil stabilizers on unpaved roads. Perma-ZymeTM  is one of the soil stabilization products selected by CERF/IIEC for evaluation according to the contract requirement. This evaluation includes environmental/toxicological tests, field demonstration and performance evaluation, and long-term performance investigation.

In May 2002, CERF/IIEC and RTI International in Research Triangle Park, NC prepared the Generic Protocol for Dust Suppression and Soil Stabilization Products as part of the work for the U.S. Environmental Protection Agency’s (EPA) Environmental Technology Verification (ETV) Program. The protocol has been approved by EPA, and evaluation of Perma-ZymeTM is conducted according to this protocol. This report is an analytical summary and theoretical calculation for Perma-ZymeTMs  impacts to environment and nearby ecological system, based on the environmental composition and toxicology analysis from state’s or EPA licensed laboratories.

CERF/IIEC is a global not- for-profit 501(c)(3) organization, created by the American Society of Civil Engineers (ASCE), focused on constructing an efficient and renewable future. In collaboration with the construction, engineering, and environmental industries, CERF/IIEC promotes and facilitates the advancement of innovation for a sustainable infrastructure. In particular, CERF/IIEC operates innovative technology programs to speed the use of innovation into practice in the areas of transportation, public works, energy systems and applications, and the environment.

2. Laboratory Information

The environmental composition was analyzed at Tri-State Laboratories, Inc. in
Youngstown, OH. Tri-State Laboratories is qualified for environmental analysis, and has Certificates of Approval from Ohio Environmental Protection Agency (OhioEPA) (Attachment A).

The toxicological analysis was conducted by ABC Laboratories in Columbia, MO. ABC Laboratories is qualified for toxicological analysis, and has the inspection certificates from US EPA, US Department of Agriculture (USDA), and US Food & Drug Administration (FDA). (Attachment B).

3. Manufacturing and Usage of Perma -ZymeTM

According to the manufacturer International Enzymes Inc., Perma-ZymeTM is formulated with enzyme-rich materials that are manufactured through a natural fermentation process using cane sugars and other natural organic compounds. When mixed with water and applied during compaction, Perma-ZymeTM acts upon the soil's organic fines through a catalytic bonding process producing a strong cementation effect. The result is a durable and water-resistant mix that can be used in any climatic environment as a sub-base or as a primary surface.

Before applied into the soil, Perma-ZymeTM is diluted with water. The dilution rate is 1:1,750 (v/v), but it may vary according to the soil moisture. Since the specific gravity ofPerma-ZymeTM  is 1.4146, the Perma-ZymeTM concentration in the application mixture is approximately 810 mg/L if the dilution rate is 1:1,750 (v/v). The Perma-ZymeTM Application Mixture will be sprayed onto the soil, which has been treated with water to get the desired moisture. The soil is mixed with Perma-ZymeTM, windrowed and compact. The final application of Perma-ZymeTM  is a gallon for every 165 cubic yard of soil. Most of the soil’s specific gravities range from 2.6 – 2.8, the Perma-ZymeTM concentration in treated soil is about 17.3 mg/kg.

4. Physical Properties of Perma-ZymeTM

Perma-ZymeTM  is a brown, non-corrosive, non- flammable, viscous fluid with slight sweet smell. The physical properties of Perma-ZymeTM are summarized in Table 4-1.

Table 4-1. Physical Properties of Perma-ZymeTM

Color 

Smell

Flammability

Corrosivity

Specific Gravity

Brown

Slight sweet smell

Non-flammable

No corrosive

1.4146

 

5. Environmental Composition Results
According to the manufacturing information provided by International Enzyme, Perma-ZymeTM is made of organic materials. The environmental composition analysis consists organic content analyses and toxic chemical analyses. The organic content analytical items are biological oxygen demand (BOD) and chemical oxygen demand (COD). The toxic chemical analyses include metals, herbicides/pesticides, polynuclear aromatic hydrocarbons (PAH), semi-volatile organic compounds (SVOC), and volatile organic compounds (VOC).

Table 5-1 through Table 5-6 show the concentrations of each item in pure Perma-ZymeTM and soil after a standard application. The concentrations in pure Perma-ZymeTM are directly from the laboratory analytical data. The concentrations in soil are calculated based on the 17.3 mg/kg of Perma-ZymeTM in soil.

Table 5-1 Organic Contents in Different Media

Analyte

EPA Method

Perma-ZymeTM(mg/L)

Soil (mg/kg)

BOD

405.1

600,000

7.34

COD

410.4

1,100,000

13.45

 

Table 5-2 Metal Concentrations in Different Media

Analyte

EPA Method

Perma-ZymeTM (mg/kg)

Soil (mg/kg)

Aluminum

200.7

2.4

0.0415

Antimony

200.7

ND

ND

Arsenic

200.7

ND

ND

Barium

200.7

2.71

0.0469

Beryllium

200.7

ND

ND

Cadmium

200.7

ND

ND

Chromium

200.7

ND

ND

Copper

200.7

ND

ND

Iron

200.7

12.6

0.2180

Lead

200.7

ND

ND

Manganese

200.7

1.35

0.0234

Mercury

200.7

0.003

0.0001

Nickel

200.7

ND

ND

Selenium

200.7

ND

ND

Silver

200.7

ND

ND

Thallium

200.7

ND

ND

Zinc

200.7

2.85

0.0493

Note: ND – non-detected

Table 5-3 Herbicides/Pesticides Contents in Different Media

Analyte

EPA Method

Perma-ZymeTM (mg/kg)

Soil (mg/kg)

2,4-D

8270

ND

ND

Silver

8270

ND

ND

Technical chlordane

8270

ND

ND

Endrin

8270

ND

ND

Heptachlor

8270

ND

ND

Lindane

8270

ND

ND

Methoxychlor

8270

ND

ND

Toxaphene

8270

ND

ND

Note: ND – non-detected

Table 5-4 PAH Contents in Different Media

Analyte

EPA Method

Perma-ZymeTM (mg/kg)

Soil (mg/kg)

Acenaphthlene

8270

ND

ND

Acenaphthylene

8270

ND

ND

Anthracene

8270

ND

ND

Bezo[a]anthracene

8270

ND

ND

Bezo[a]pyrene

8270

ND

ND

Bezo[b]fluoranthene

8270

ND

ND

Benzo[g,h,I]perylene

8270

ND

ND

Benzo[k]fluoranthene

8270

ND

ND

Chrysene

8270

ND

ND

Dibenzo[a,h]anthracene

8270

ND

ND

Fluoranthene

8270

ND

ND

Fluorene

8270

ND

ND

Indeno(1,2,3-cd)pyrene

8270

ND

ND

Naphthalene

8270

ND

ND

Phenathrene

8270

ND

ND

Pyrene

8270

ND

ND

Note: ND – non-detected

 

Table 5-5 SVOC Contents in Different Media

Analyte

EPA Method

Perma-ZymeTM (mg/kg)

Soil (mg/kg)

Acenapthene

8270

ND

ND

Acenaphthylene

8270

ND

ND

Anthracene

8270

ND

ND

Benzidine

8270

ND

ND

Benzo [a] anthracene

8270

ND

ND

Benzo [a] pyrene

8270

ND

ND

3,4-Benzofluoranthene

8270

ND

ND

Benzo (g,h,I) perylene

8270

ND

ND

Benzo (b) fluoranthene

8270

ND

ND

Benzo (k) fluoranthene

8270

ND

ND

Bis (2-chloroethoxy)
methane

8270

ND

ND

Bis (2-chloroethyl) ether

8270

ND

ND

Bis (2-chloroisopropyl)
ether

8270

ND

ND

Bis (2-ethylhexyl) phthalate

8270

ND

ND

4-Bromophenyl phenyl ether

8270

ND

ND

Butyl benzyl phthalate

8270

ND

ND

2-Chloronaphthalene

8270

ND

ND

4-Chlorophenyl phenyl ether

8270

ND

ND

Chrysene

8270

ND

ND

Dibenzo [a,h] anthracene

8270

ND

ND

1,2-Dichlorobenzene

8270

ND

ND

1,3-Dichlorobenzene

8270

ND

ND

1,4-Dichlorobenzene

8270

ND

ND

3,3’-Dichlorobenzidine

8270

ND

ND

Diethyl phthalate

8270

ND

ND

 

Table 5-5 SVOC Contents in Different Media (Continuing)

Dimethyl phthalate

8270

ND

ND

Di-n-butyl phthalate

8270

ND

ND

2,4-Dinitrotoluene

8270

ND

ND

2,6-Dinitrotoluene

8270

ND

ND

Di-n-octyl phthalate

8270

ND

ND

1.2-Diphenylhydrazine (as azobenzene)

8270

ND

ND

Fluoranthene

8270

ND

ND

Fluorene

8270

ND

ND

Hexachlorobenzene

8270

ND

ND

Hexachlorobutadiene

8270

ND

ND

Hexachlorocyclopentadiene

8270

ND

ND

Hexachloroethane

8270

ND

ND

Indeno (1,2,3-cd)pyrene

8270

ND

ND

Isophorone

8270

ND

ND

Naphthalene

8270

ND

ND

Nitrobenzene

8270

ND

ND

N-Nitrosodimethylamine (as diphenylamine)

8270

ND

ND

N-Nitrosodi-n-propylamine

8270

ND

ND

N-Nitrosodiphenylamine

8270

ND

ND

Phenanthrene

8270

ND

ND

Pyrene

8270

ND

ND

1,2,4-Trichlorobenzene

8270

ND

ND

2-Chlorophenol

8270

ND

ND

2,4-Dichlorophenol

8270

ND

ND

2,4-Dimethylphenol

8270

ND

ND

4,6-Dinitro-o-cresol

8270

ND

ND

2,4-Dinitrophenol

8270

ND

ND

2-Methyl phenol

8270

ND

ND

3&4-Methyl phenol

8270

ND

ND

2-Nitrophenol

8270

ND

ND

4-Nitrophenol

8270

ND

ND

Pentachlorophenol

8270

ND

ND

Phenol

8270

ND

ND

2,4,5- Trichlorophenol

8270

ND

ND

2,4,6- Trichlorophenol

8270

ND

ND

Note: ND – non-detected

Table 5-6 VOC Contents in Different Media

Analyte

EPA Method

Perma-ZymeTM  (mg/kg)

Soil (mg/kg)

Acetone

8260

ND

ND

Benzene

8260

ND

ND

Bromobenzene

8260

ND

ND

Bromochloromethane

8260

ND

ND

Bromodichloromethane

8260

ND

ND

Bromoform

8260

ND

ND

Bromomethane

8260

ND

ND

2-Butanone

8260

10,674

184.7

n-Butylbenzene

8260

ND

ND

sec-Butylbenzene

8260

ND

ND

tert-Butylbenzene

8260

ND

ND

Carbon Tetrachloride

8260

ND

ND

Chlorobenzene

8260

ND

ND

Chloroethane

8260

ND

ND

Chloroform

8260

ND

ND

Chloromethane

8260

ND

ND

2-Chlorotoluene

8260

ND

ND

4-Ch’orotoluene

8260

ND

ND

1,2-Dibromo-3-
chloropropane

8260

ND

ND

Dibromochloromethane

8260

ND

ND

1 ,2-Dibromoethane

8260

ND

ND

Dibromomethane

8260

ND

ND

1,2-Dichlorobenzene

8260

ND

ND

1,3-Dichlorobenzene

8260

ND

ND

1,4-Dichlorobenzene

8260

ND

ND

Dichlorodifluoromethane

8260

ND

ND

1, 1-Dichloroethane

8260

ND

ND

1,2-Dichloroethane

8260

ND

ND

1,1,-Dichloroethene

8260

ND

ND

cis-1 ,2-Dichloroethene

8260

ND

ND

trans-1,2-Dichloroethene

8260

ND

ND

1,2-Dichloropropane

8260

ND

ND

1,3-Dichloropropane

8260

ND

ND

2,2-Dichloropropane

8260

ND

ND

1, 1-Dichloropropene

8260

ND

ND

Ethyl Benzene

8260

ND

ND

Hexachlorobutadiene

8260

ND

ND

2-Hexanone

8260

ND

ND

Isopropylbenzene

8260

ND

ND

p-lsopropyltoluene

8260

ND

ND

Methylene Chloride

8260

ND

ND

Methyl Isobutyl Ketone

8260

ND

ND

Naphthalene

8260

ND

ND

n-Propylbenzene

8260

ND

ND

Styrene

8260

ND

ND

1,1,1,2-
Tetrachloroethane

8260

ND

ND

 

Table 5-6 VOC Contents in Different Media (Continuing)

1,1,2,2-
Tetrachloroethane

8260

ND

ND

Tetrachloroethene

8260

ND

ND

Toluene

8260

ND

ND

1,2,3- Trichlorobenzene

8260

ND

ND

1,2,4- Trichlorobenzene

8260

ND

ND

1,1 ,1- Trichloroethane

8260

ND

ND

1,1,2- Trichloroethane

8260

ND

ND

Trichloroethene

8260

ND

ND

Trichlorofluoromethane

8260

ND

ND

1,2,3- Trichloropropane

8260

ND

ND

1,2,4-
Trimethylbenzene

8260

ND

ND

1,3,5-
Trimethylbenzene

8260

ND

ND

Vinyl Chloride

8260

ND

ND

m,p-Xylene

8260

ND

ND

o-Xylene

8260

ND

ND

Note: ND – non-detected

The above tables indicate that seven chemicals and BOD/COD were detected in Perma-ZymeTM. The chemical concentrations in soil are compared with Risk-Based Concentrations (RBC) in residential soil, which was developed by US EPA as a screen level for contaminants on a concerned site. Table 5-7 compares RBC values and chemical concentrations in soil caused by Perma-ZymeTM  application.

Table 5-7 RBC Values and Soil Concentrations

Chemical

RBC Level in Residential Soil (mg/kg)

Chemical in Soil (mg/kg)

BOD

N/A

7.34

COD

N/A

13.45

Aluminum

48,000

0.0415

Barium

5,500

0.0469

Iron

23,000

0.2180

Manganese

1,600

0.0234

Mercury

7.8

0.0001

Zinc

23,000

0.0493

2-Butanone

47,000

184.7

BOD/COD are two major indicators for organic component contents. The BOD/COD values are relative high in the soil due to the fact that Perma-ZymeTM is made from cane sugar. During the manufacturing of Perma-ZymeTM, cane sugar is used as raw material and goes through fermentation. The additives are added to the fermentation products, which produces Perma-ZymeTM.

From Table 5-7, it can be found that all detected chemical concentrations are significant lower compared with the RBC levels in residential soil, which means that application of Perma-ZymeTM  does not increase risk level of soils. Therefore, Perma-ZymeTM  is safe for use in the term of environmental impact.

6. Toxicological Analysis Results

In this particular toxicological test, LC50 is used in the laboratory to indicate the toxicity of pure Perma-ZymeTM  in water, according to Method EPA/600/4-90/027F and EPA/600/4-91/002 for acute and chronic toxicity, respectively. LC50 means lethal concentration in water that kills 50% of small aquatic creatures used in the test. The toxicity analytical results are listed in Table 6-1.

Table 6-1 Toxicity Analytical Results

Pure Perma-
ZymeTM
Concentration
(mg/L)

Percent Mortality

Ceriodaphnia Dubia

Fathead Minnow

Americamysis Bahia

Acute

Chronic

Acute

Chronic

Acute

Chronic

Control

0

0

0

3

0

15

0.13

-

-

-

-

-

17

0.25

-

-

-

-

-

25

0.5

-

-

-

-

-

15

1.0

-

-

-

-

0

10

1.3

0

0

0

0

-

-

2.0

-

-

-

-

65

35

2.5

0

0

0

3

-

-

4.0

-

-

-

-

90

-

5.0

-

0

5

13

-

-

8.0

-

-

-

-

100

-

10.0

0

80

35

100

-

-

16.0

-

-

-

-

100

-

20.0

80

100

100

100

-

-

40.0

100

-

-

-

-

-

According to EPA-540-9-85-006, suggested toxicity criteria for materials are listed in Table 6-2.

Table 6-2 Toxicity Categories

LC50 (mg/L)

Category Description

<0.1

Very highly toxic

0.1 – 1

Highly toxic

1 – 10

Moderately toxic

10 –100

Slightly toxic

>100

Practically non-toxic

If 100 mg Perma-ZymeTM Application Mixture (containing 810 mg/L pure Perma-
ZymeTM) is diluted with 1 liter control solution, it generates 100 mg/L of Perma-ZymeTM Application Mixture solution. This solution contains 0.081 mg/L pure Perma-ZymeTM. From Table 6-1, it can be found that all the toxicity analysis results will be less than 50% at the Perma-ZymeTM concentration of 0.081 mg/L; therefore, the LC50 values for Perma- ZymeTM Application Mixture should be greater than 100 mg/L. The LC50 values and toxicity categories for pure Perma-ZymeTM and Perma-ZymeTM Application Mixture are summaries in Table 5-3.

Table 6-3 LC50 and Toxicity Category

 

Pure Perma-ZymeTM

Perma-ZymeTM Application Mixture

LC50
(mg/L)

Toxicity Category

LC50
(mg/L)

Toxicity Category

Ceriodaphnia Dubia acute

16.2

Slightly toxic

>100

Practically non-toxic

Ceriodaphnia Dubia chronic

8.1

Moderately toxic

>100

Practically non-toxic

Fathead Minnow acute

6.4

Slightly toxic

>100

Practically non-toxic

Fathead Minnow chronic

6.4

Moderately toxic

>100

Practically non-toxic

Americamysis Bahia acute

1.7

Moderately toxic

>100

Practically non-toxic

Americamysis Bahia chronic

>2

Moderately toxic

>100

Practically non-toxic

From Table 6-3, it can be found that pure Perma-ZymeTM is either slightly toxic or
moderately toxic in different testing methods. While, Perma-ZymeTM  Application
Mixture is practically non-toxic in all cases.

7. Conclusion

Based on the calculation and discussion in Sections 5 and 6, the following conclusions can be drawn:

 

Attachment A
Certificates of Approval of Tri-State Laboratories

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Attachment B
Qualification of ABC Laboratories

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