CELLULASE ENZYME
This dossier on cellulase enzyme presents the most critical studies pertinent to the risk assessment of cellulase enzyme in its use in hydraulic fracturing fluids. It does not represent an exhaustive or critical review of all available data. Where possible, study quality was evaluated using the Klimisch scoring system (Klimisch et al., 1997).
Screening Assessment Conclusion – Cellulase enzyme is classified as a tier 1 chemical and requires a hazard assessment only.
1 BACKGROUND
Cellulase enzymes are catalytic proteins or polypeptides that consist of amino acids coupled via peptide bonds. Cellulase enzymes cleave ß-1,4-glycosidic bonds in cellulose. Cellulases are readily biodegradable; they are not expected to bioaccumulate or adsorb to soil. Cellulase enzyme has a moderate acute toxicity concern to aquatic organisms.
2 CHEMICAL NAME AND IDENTIFICATION
Chemical Name (IUPAC): 1,4-(1,3;1,4)-β-D-glucan-4-glucanohydrolase
CAS RN: 9012-54-8
Molecular weight: 20,000 to 80,000 Daltons
Synonyms: Cellulase; 1,4-(1,3;1,4)-β-D-glucan-4-glucanohydrolase;
Enzymes are catalytic proteins or polypeptides that consist of amino acids coupled via peptide bonds. There is a broad range of cellulases used commercially that come from fungal and bacterial origins; all are characterised by ß-1,4-endoglucanase activity. Cellulase enzymes cleave ß-1,4- glycosidic bonds in cellulose.
Enzyme preparations are characterised by their enzymatic activity according to the specific methods of the producer. To compare different enzyme preparations, the amount of active substance is normally calculated from the activity via the specific activity of the enzyme, where the protein is determined by active site titration and/or quantitative and qualitative amino acid analysis. The resulting active enzyme protein (aep) content represents a value based on a theoretical pure and totally active enzyme (HERA, 2005).
3 PHYSICO-CHEMICAL PROPERTIES
Key physical and chemical properties for the substance are shown in Table 1. Table 1 Overview of the Physico-chemical Properties of Cellulase
Property Value Klimisch score Reference Physical state at 20oC and 101.3 kPa As pure enzyme, white crystals or - HERA, powder 2005
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Property Value Klimisch score Reference Melting Point Not feasible - ECHA Boiling Point Not feasible - ECHA Density >1330 - <1420 kg/m3 @ 20°C 2 ECHA Vapour Pressure 0.00344 Pa @ 25°C (mean value) 1 ECHA
Partition Coefficient (log Kow) < -1.3 @ 20°C* 1 ECHA Water Solubility 100 g/L @ 25 °C - ECHA
*An octanol-water partition coefficient (Kow) for cellulases is not available. Therefore, the log Kow of glucoamylase, which cleaves 1,4–α-D-glycosidic linkages, was referenced.
4 DOMESTIC AND INTERNATIONAL REGULATORY INFORMATION
A review of international and national environmental regulatory information was undertaken (Table 1). This chemical is listed on the Australian Inventory of Chemical Substances – AICS (Inventory). No conditions for its use were identified. No specific environmental regulatory controls or concerns were identified within Australia and internationally for cellulase enzyme.
Based on an assessment of environmental hazards, NICNAS identified cellulase enzyme as a chemical of low concern to the environment (NICNAS, 2017). Chemicals of low concern are unlikely to have adverse environmental effects if they are released to the environment from coal seam gas operations. Table 1 Existing International Controls
Convention, Protocol or other international control Listed Yes or No? Montreal Protocol No Synthetic Greenhouse Gases (SGG) No Rotterdam Convention No Stockholm Convention No REACH (Substances of Very High Concern) No United States Endocrine Disrupter Screening Program No European Commission Endocrine Disruptors Strategy No
5 ENVIRONMENTAL FATE SUMMARY
A. Summary
Cellulases are readily biodegradable; they are not expected to bioaccumulate or adsorb to soil.
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B. Biodegradation
A cellulase enzyme was readily biodegradable in an OECD 301F test. There was ~10% degradation after one day; ~60% after 5 days; and 129% after 28 days (ECHA). [Kl. score = 1]
Three different cellulase enzymes were considered readily biodegradable based on the results of OECD 301C and 301E tests (HERA, 2005) [Kl. scores = 1]. In an OECD 301E test, there was 84% DOC removal of the enzyme Carezyme after 28 days. In another OECD 301E test, there was 92% DOC removal of the enzyme Clazinase® after 28 days. In an OECD 301C test, BOD/COD was 78% after 28 days (HERA, 2005).
If a chemical is found to be readily biodegradable, it is categorised as Not Persistent since its half-life is substantially less than 60 days (DoEE, 2017).
C. Environmental Distribution
Proteins, such as cellulase enzyme, would not be expected to adsorb to soil. HERA (2005) listed a Koc value of <1.3 for detergent amylases, cellulases, and lipases that was calculated according to the EU Technical Guidance Document (EC, 2003). No further information was provided. If released to water, based on its low Koc and high water solubility values, cellulase is likely to remain in water and not adsorb to sediment. It is also not expected to adsorb to soil, and, has the potential to be highly mobile.
D. Bioaccumulation
No bioaccumulation studies have been conducted on cellulase enzymes. Cellulase enzymes are not expected to bioaccumulate due to their high molecular weight, high water solubility and their low log Kow (log Kow is <-1.3). Moreover, cellulases are rapidly biotransformed in an organism to lower molecular-weight protein fragments by proteolytic enzymes (proteases), and eventually to the basic amino acids by peptidase enzymes.
6 ENVIRONMENTAL EFFECTS SUMMARY
A. Summary
Cellulase enzyme has a moderate acute toxicity concern to aquatic organisms.
B. Aquatic Toxicity
Acute Studies
Table 2 lists the results of acute aquatic toxicity studies on cellulase enzyme. Table 2 Acute Aquatic Toxicity Studies on Cellulase Enzyme
Test Species Endpoint Results (mg/L) Klimisch Reference score
Oncorhynchus mykiss 96-hr LC50 >100* 1 ECHA >52.1**
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Brachydanio rerio 96-hr LC50 330* 4 HERA (2005)
Daphnia magna 48-hr EC50 >100* 1 ECHA >52.1**
Daphnia magna 48-hr EC50 >1,000** 4 HERA (2005)
Daphnia magna 48-hr EC50 1,000** 4 HERA (2005)
Pseudokirchneriella 72-hr EC50 >100* 1 ECHA subcapitata >52.1**
Scenedesmus subspicatus 72-hr EC50 >1,000** 4 HERA (2005)
*Total organic solids. **Active enzyme protein.
Chronic Studies
No studies are available.
C. Terrestrial Toxicity
No studies are available.
7 CATEGORISATION AND OTHER CHARACTERISTICS OF CONCERN
A. PBT Categorisation
The methodology for the Persistent, Bioaccumulative and Toxic (PBT) substances assessment is based on the Australian and EU REACH Criteria methodology (DEWHA, 2009; ECHA, 2008).
Cellulase enzyme is readily biodegradable; thus it does not meet the screening criteria for persistence.
Cellulase enzyme has a high molecular weight (20 to 80 kilo Daltons-kD), hydrophilic properties (high water solubility, log Kow <-1.3), and is readily biotransformed in organisms by proteases and peptidases. Thus cellulase enzyme does not meet the screening criteria for bioaccumulation.
There are no chronic toxicity studies on cellulase enzyme. The acute EC50 values are >1 mg/L in fish, invertebrates and algae. Thus cellulase enzyme does not meet the criteria for toxicity.
The overall conclusion is that cellulase enzyme is not a PBT substances.
B. Other Characteristics of Concern
No other characteristics of concern were identified for cellulase enzyme.
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8 SCREENING ASSESSMENT
Chemical Databases of Concern Persistence Assessment Bioaccumulative Toxicity Assessment Step Assessment Step Step Assessment Step Overall PBT Risk Assessment Chemical Name CAS No. Listed as a COC Identified as Assessment 1 P criteria Other P T criteria Acute Chronic Actions Required3 on relevant Polymer of Low B criteria fulfilled? fulfilled? Concerns fulfilled? Toxicity 2 Toxicity2 databases? Concern
Cellulase enzyme 9012-54-8 Not a PBT No No No No No No 1 No data 1 Footnotes:
1 - PBT Assessment based on PBT Framework. 2 - Acute and chronic aquatic toxicity evaluated consistent with assessment criteria (see Framework). 3 – Tier 1 – Hazard Assessment only. Notes:
PBT = Persistent, Bioaccumulative and Toxic
B = bioaccumulative
P = persistent
T = toxic
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9 REFERENCES, ABBREVIATIONS AND ACRONYMS
A. References
Department of the Environment, Water, Heritage and the Arts [DEWHA]. (2009). Environmental risk assessment guidance manual for industrial chemicals, Department of the Environment, Water, Heritage and the Arts, Commonwealth of Australia.
Department of the Environment and Energy [DoEE]. (2017). Chemical Risk Assessment Guidance Manual: for chemicals associated with coal seam gas extraction, Guidance manual prepared by Hydrobiology and ToxConsult Pty Ltd for the Department of the Environment and Energy, Commonwealth of Australia, Canberra.
ECHA. ECHA REACH database: http://echa.europa.eu/information-on-chemicals/registered- substances
European Commission [EC]. (2003). Technical Guidance Document on Risk Assessment. Part II. Environmental Risk Assessment.
European Chemicals Agency [ECHA]. (2008). Guidance on Information Requirements and Chemical Safety Assessment, Chapter R11: PBT Assessment, European Chemicals Agency, Helsinki, Finland.
HERA. (2005). Human and Environmental Risk Assessment (HERA) on Ingredients of Household Cleaning Products: α-amylase, CAS No. 9000-90-2; Cellulase (β-(1,4)- glucanase), CAS No. 9012-54-8; Lipase, 9001-62-1, 2005. http://www.heraproject.com.
Klimisch, H.J., Andreae, M., and Tillmann, U. (1997). A systematic approach for evaluating the quality of experimental and toxicological and ecotoxicological data. Regul. Toxicol. Pharmacol. 25:1-5.
NICNAS. (2017). National assessment of chemicals associated with coal seam gas extraction in Australia,Technical report number 14 - Environmental risks associated with surface handling of chemicals used in coal seam gas extraction in Australia. Project report prepared by the Chemicals and Biotechnology Assessments Section (CBAS), in the Chemicals and Waste Branch of the Department of the Environment and Energy as part of the National Assessment of Chemicals Associated with Coal Seam Gas Extraction in Australia, Commonwealth of Australia, Canberra.
B. Abbreviations and Acronyms
°C degrees Celsius AICS Australian Inventory of Chemical Substances BOD biological oxygen demand COC constituent of concern COD chemical oxygen demand
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DEWHA Department of the Environment, Water, Heritage and the Arts DOC dissolved organic carbon EC effective concentration ECHA European Chemicals Agency EU European Union IUPAC International Union of Pure and Applied Chemistry kD kilo Daltons kg/m3 kilograms per cubic metre Kl Klimisch scoring system LC lethal concentration mg/L milligrams per litre OECD Organisation for Economic Co-operation and Development PBT Persistent, Bioaccumulative and Toxic REACH Registration, Evaluation, Authorisation and Restriction of Chemicals SGG Synthetic Greenhouse Gases
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