Page 2 of 12

Executive Summary 

Dibromodicyanobutane (DBDCB) is an organo-cyanobromine compound registered for 

use as an algicide, bactericide, fungicide, slimicide, and microbicide/microbistat in industrial 

process and water systems such as gas/oil drilling muds/packer fluids, completion and workover 

fluids, water floods, gas/oil recovery injection systems, enhanced oil recovery systems, oil field 

water, polymer of micellar floods, water-disposal systems or other oil field water systems, 

cooling water tower systems, paper mills, pulp, paper and paperboard systems and as a material 

preservative. Pesticide products containing the active ingredient DBDCB were first registered in 

the United States in 1980. Currently there are 16 registered products with active ingredient 

declarations of 23.725 to 99.9 percent. All product chemistry data requirements have been 

satisfied for this active ingredient DBDCB. 

 The submitted environmental fate studies demonstrate that parent DBDCB is generally 

hydrolytically less persistent than its degradates, trans 1-bromo-2,4-dicyanobutene (BDCB, 

once-debrominated parent) and 2-methyleneglutaronitrile (MGN, twice debrominated parent), 

under alkaline conditions. They also demonstrate that the degradate BDCB (trace levels present 

only) biodegrades aerobically faster than the parent (half-life 0.87 days) or MGN (4.6 days). 

Given that all residues of DBDCB and its major degradates have limited sorption to soil and 

sediment, the primary residues that can reach surface and ground water are DBDCB and its 

degradate MGN. The overall degradation of DBDCB appears to lead to stable degradates that 

have not been identified, but the remaining portion of the compound is a straight-chain aliphatic 

hydrocarbon which can become part of the organic matter of soil or biosolids. The registrant has 

submitted environmental fate studies that were considered to be “supplemental, upgradeable” 

and “not completely acceptable”. However, the information is of sufficient quality to use in the 

ecological risk assessment. To address fate of the more stable degradate MGN through the waste 

water treatment plant process, biodegradability in sewage sludge is needed and an activated 

sludge respiration inhibition study are anticipated. Based on the environmental fate information, 

drinking water consumption from ground or surface water is likely to be limited for parent 

DBDCB but is more likely for the metabolite MGN. 

The aquatic organism toxicity data base for DBDCB contains acute and chronic 

freshwater fish and invertebrate data and acute estuarine/marine fish and invertebrate data for 

DBDCB and avian acute oral and subacute dietary data. DBDCB is moderately toxic to 

freshwater and estuarine/marine animals and slightly toxic to birds and mammals. In order to 

perform an ecological risk assessment, including an endangered species determination, the 

Agency anticipates needing algal (Tier II) toxicity studies using freshwater green alga, 

freshwater diatom, marine diatom, and cyanobacterium, and; an aquatic plant toxicity study 

using Lemna spp. If the IC50 is >1000 ppb a.i. for freshwater green alga, then freshwater diatom, 

marine diatom, cyanobacterium and aquatic plant toxicity studies can be waived. No toxicity 

data is available for the MGN degradate, toxicity of this degradate will be assumed to be as toxic 

as the parent. 
 Page 3 of 12

Introduction 

DBDCB was first registered in the United States in 1980. Currently 16 products are 

registered for use as an algicide, bactericide, fungicide, slimicide, and microbicide/microbistat in 

industrial process and water systems such as gas/oil drilling muds/packer fluids, completion and 

workover fluids, water floods, gas/oil recovery injection systems, enhanced oil recovery systems, 

oil field water, polymer of micellar floods, water-disposal systems or other oil field water 

systems, cooling water tower systems, paper mills, pulp, paper and paperboard systems and as a 

material preservative in such products as adhesives, glues and tackifiers (nonfood contact), 

aqueous paints and coatings, dispersed pigments, latex emulsions, paper and paper additives, 

paints, coatings and stains, metalworking cutting fluids, fiber processing fluids, joint cement, as 

well as dishwashing liquids, furniture and floor waxes and polishes, cleaners and treatment 

products and over-the counter inks. A Reregistration Eligibility Decision (RED) was issued by 

EPA for DBDCB in June 19961

. 

A. Product Chemistry Summary of DBDCB (99.9% TGAI) 

Common Name: Dibromodicyanobutane 

Methyldibromoglutaronitrile

Bromothalonil

Chemical Name: 1-bromo-(bromoethyl)-1,3-propanedicarbonitrile

1,2-dibromo-2,4-dicyanobutane 

1,2-dibromo-2,4-dicyanobutane (dibromodicyanobutane)

Pentanedinitrile, 2-bromo-2-(bromomethyl)

Trade and Other Names: Tektamer 38; Metacide 38; Merck 48051 

1-Bromo-1-(bromomethyl)-1,3-propanedicarbonitrile 

2-Bromo-2-(bromomethyl)glutaronitrile 

2-Bromo-2-(bromomethyl)pentanedinitrile 

Merguard 1105 

OPP Chemical Code: 111001 

CAS Registry Number: 35691-65-7 

Molecular Formula: C6H6Br2N2

Molecular weight: 265.9340 g/mole 

Molecular Structure: 

 Color: Yellowish white; off-white to light tan. 

Physical State: Granular solid at 21.72°C; crystalline solid 

 

1

http://www.epa.gov/oppsrrd1/REDs/2780red.pdf 
 Page 4 of 12

Odor: Slightly sweet at room temperature; mildly pungent. 

Stability: The chemical was found to be stable when subjected to 

aluminum, tin, nickel, iron, thermal and photosensitivity 

tests. Stable for one year at room temperature.

pH: 4.9 

UV/Visible absorption: Maximum at 290 nm. No UV absorbance above 290 nm.

Melting point: 51-53°C 

Density: 0.970 g/ml at 24.28°C 

Dissociation Constant: Not applicable. Chemical does not ionize. 

Log Ko/w: 1.022 

Solubilities: Water: 0.212 g/100 ml at 20°C 

Organic solvents: It is soluble in diethyl ether, ethanol, and 

methanol and very soluble in acetone, benzene, chloroform, 

dimethylformamide, ethyl acetate. 

Vapor Pressure: 5.0 × 10-5 mm Hg at 25°C 

Henry's Law Constant: 8.25 x 10-9 atm-m3

/mole (Calculated) 

Enforcement Analytical Mass Spectrometry; Infra-red spectrometry

Method: Gas Chromatography 

All product chemistry data requirements have been satisfied for the active ingredients 

DBDCB (Source: RED). 

B. Environmental Fate Summary 

 The registrant has submitted environmental fate studies that were considered to be 

“supplemental, upgradeable” and “not completely acceptable”. However, there are no 

environmental fate data gaps for DBDCB. The dissipation of DBDCB should be controlled by 

rapid alkaline-catalyzed hydrolysis and microbial-mediated degradation in aquatic systems. The 

overall degradation appears to lead to stable degradates that have not been identified, but the 

remaining portion of the compound is a straight-chain aliphatic hydrocarbon which can become 

part of the organic matter of soil. The parent compound is very mobile in soil with Freundlich 

Kads values of <1. 

 The hydrolysis (MRID 00055032) and aerobic aquatic metabolism (MRID 41883902) 

studies both demonstrated degradation of parent DBDCB. In the hydrolysis study, the half-lives 

at 25 o

C were 190, 24, and 13 days at pH 5, 7, and 9, respectively. The only degradate that 

reached significant (>10 % of applied parent) concentrations at pH 5 and 7 was oncedebrominated parent compound (BDCB, trans 1-bromo-2,4-dicyanobutene), which increased 

until the end of the study. The degradate MGN (2-methylenegluaronitrile, twice-debrominated 

parent compound) reached significant concentrations at pH 9 and increased until the end of the 

study. In the aerobic aquatic metabolism study, the total system half-life of parent DBDCB was 

0.87 days, and the only significant degradate was MGN which reached up to 34 % of applied 

parent compound by 3 days, and declined to 0.1 % by 30 days (end of study). MGN biodegraded 

with a half-life of 4.6 days. Carbon dioxide (CO2) was also formed. Table 1 below contains a 

summary table of the environmental fate of parent DBDCB and its primary degradates. 
 Page 5 of 12

 The leaching-adsorption-desorption study was also considered to contain “supplemental, 

upgradeable” information because the desorption was greater than the adsorption. However, 

these issues do not invalidate the studies because the terminal significant degradates (cis and 

trans isomers of parent compound minus a bromine) are more water soluble and sorbed to a 

lower degree. Radiolabeled DBDCB, at 0.1 to 1.0 μg/ml, had Freundlich coefficients of 0.26 

(Koc=528; Freundlich 1/n=1.33) in a sand, 0.35 (Koc=88; 1/n=1.05) in a sandy loam soil, 0.47 

(Koc=73; 1/n=0.99) in a clay loam soil, and 0.70 (Koc=33; 1/n=1.09) in a silt loam soil. DBDCB 

degradation was observed during a 2 hour equilibration period. The cis and trans isomers of 

BDCB were detected (6-75% of applied) by high performance liquid chromatography (HPLC). 

Desorption coefficients were not reported. However, the mean percent desorption of DBDCB 

was 125% for a sand, 107% for a sandy loam soil, 148% for a clay loam soil, and 126% for a silt 

loam soil. The material balance is off because of rapid degradation to biodegradation products 

with less sorption than the parent compound. The reported data indicate DBDCB should be 

mobile in aquatic and soil environments. Based on the above environmental fate information, 

drinking water consumption from ground or surface water is likely to be limited for parent 

DBDCB but is more likely for the MGN biodegradate. 

 To address fate of DBDCB and its MGN degradate through the waste water treatment 

plant process, biodegradability in sewage sludge is needed and an activated sludge respiration 

inhibition study are anticipated. 

Table 1. Summary Table of Environmental Fate Data for DBDCB and Degradates

Parameters 

Parent Degradates 

Source/ 

Comments Dibromodicyanobutane (DBDCB) 

trans 1-bromo-2,4-

dicyanobutene (BDCB) 

(parent compound minus 

one bromine) 

2-methylene 

glutaronitrile (MGN) 

(parent compound 

minus two bromines) 

Structures 41883901 

Dissociation 

Constant (pKa) 

Not applicable, 

chemical does not 

ionize 

Not applicable, chemical 

does not ionize 

Not applicable, 

chemical does not ionize 

Hydrolysis halflife (835.2120)

pH 5, 190 days 

pH 7, 21 days 

pH 9, 13 days 

Slowly degrades1

Significant (>10 % of 

applied), increased until 

end of study; terminal 

significant degradate. 

Slowly degrades2

Did not reach significant 

(>10 %) concentrations 

at pH 5 and 7, but did so 

at pH 9; terminal 

significant degradate at 

pH 9. 

00055032 

(10 ppm @ 25 o

C)
 Page 6 of 12

Parameters 

Parent Degradates 

Source/ 

Comments Dibromodicyanobutane (DBDCB) 

trans 1-bromo-2,4-

dicyanobutene (BDCB) 

(parent compound minus 

one bromine) 

2-methylene 

glutaronitrile (MGN) 

(parent compound 

minus two bromines) 

Hydrolysis halflife (835.2120)

pH 5, 96 days 

pH 7, 14 days 

pH 9, 0.6 days 

Slowly degrades1

Significant (>10 % of 

applied), increased until 

end of study; terminal 

significant degradate. 

Slowly degrades2

Did not reach significant 

(>10 %) concentrations 

at pH 5 and 7, but did so 

at pH 9; terminal 

significant degradate at 

pH 9. 

00055032 

(10 ppm @ 45 o

C)

Hydrolysis halflife (835.2120)

pH 5, 198 days 

pH 7, 73 days 

pH 9, 14 days 

Slowly degrades1

Significant (>10 % of 

applied), increased until 

end of study; terminal 

significant degradate. 

Slowly degrades2

Did not reach significant 

(>10 %) concentrations 

at pH 5 and 7, but did so 

at pH 9; terminal 

significant degradate at 

pH 9. 

00055032 

(40 ppm @ 25 o

C)

Hydrolysis halflife (835.2120)

pH 5, 122 days 

pH 7, 11 days 

pH 9, 0.8 days 

Slowly degrades1

Significant (>10 % of 

applied), increased until 

end of study; terminal 

significant degradate. 

Slowly degrades2

Did not reach significant 

(>10 %) concentrations 

at pH 5 and 7, but did so 

at pH 9; terminal 

significant degradate at 

pH 9. 

00055032 

(40 ppm @ 45 o

C)

Aerobic aquatic 

metabolism halflife (835.4300) 0.87 day Trace levels detected 

(<10% of parent) 4.6 days4

 41883901 

Soil absorption 

constant, Kads 

(835.1230)

Kads values of 0.26-0.7 

ml/g 

Koc 33-528 ml/g 

Present at 6-75 % of 

applied parent compound 

by equilibrium (more 

mobile than parent).3

No data 41883902 

1

 No half-life could be calculated because it increased until the end of the study. The formation was faster than the decline, 

which defines this as a terminal degradate. 

2

 No half-life could be calculated at pH 9 because it increased until the end of the study. The formation was faster than the 

decline, which defines this as a terminal degradate. 

3 Because biodegradation was so rapid, no meaningful Kads value could be obtained. 4

 Leads to stable degradates that have not been identified, but the remaining portion of the compound is a straightchain aliphatic hydrocarbon which can become part of the organic matter of soil.

Water Quality 

DBDCB is not identified as a cause of impairment for any water bodies listed as impaired 

under section 303(d) of the Clean Water Act, based on information provided at the Agency’s 

National Pesticides Cause of Impairment of Waters website (searched March 29, 2012)2

. In 

addition, no Total Maximum Daily Loads (TMDL) have been developed for DBDCB, based on 

 

2

 http://iaspub.epa.gov/tmdl_waters10/attains_nation_cy.cause_detail_303d?p_cause_group_id=885
 Page 7 of 12

information provided at the Agency’s National Pesticide TMDL web site (searched, March 29, 

2012)3. More information on impaired water bodies and TMDLs can be found at 

http://www.epa.gov/owow/tmdl/. The Agency invites submission of water quality data for this 

pesticide. To the extent possible, data should conform to the quality standards in Appendix A of 

the OPP Standard Operating Procedure: Inclusion of Impaired Water Body and Other Water 

Quality Data in OPP’s Registration Review Risk Assessment and Management Process (see: 

http://www.epa.gov/oppfead1/cb/ppdc/2006/november06/session1-sop.pdf ), in order to ensure 

they can be used quantitatively or qualitatively in pesticide risk assessments. 

C. Ecological Effects Summary 

No quantitative ecological risk assessment was performed for DBDCB at the time of the 

reregistration. Available ecotoxicity guideline studies for DBDCB and the results are 

summarized in Table 2 below. An acute-to-chronic ratio (ACR) is 2.334

 for freshwater fish 

based on rainbow trout data and 1.575 for freshwater invertebrates based on Daphnia magna

data. In the D. magna acute and chronic high purity active ingredient studies, the acute NOAEC 

was lower than the chronic NOAEC, which may indicate that the organisms used in the acute 

study were potentially more sensitive than those used in the chronic, or that the ability to detect 

differences from the control was not as high in the chronic study as compared to the acute. The 

ACR may be underestimated to some degree in either case. There is no toxicity data for the 

major degradate MGN. This degradate will be assumed to be as toxic as the parent compound. 

Table 2. Ecotoxicity Data Summary for DBDCB 

MRID 

NOs. 

COMMON 

NAME 

TAXONOMIC 

NAME 

GUIDE

LINE TYPE % a.i. 

STUDY 

TIME

DOSE

TYPE

TOXI

CITY 

TOX 

LEVEL NOEL STUDY STATUS 

TOX 

CATEGORY/

Avian Acute Oral Toxicity

00077315 Mallard 

duck 

Anas 

platyrhynchos 

850.2100

71-1 

O Tech 14 D LD50 1038 mg/kgbw <215 Supplemental Slightly 

Toxic 

Avian Subacute Dietary Toxicity

00077317 Mallard 

duck 

Anas 

platyrhynchos 

850.2200

71-2b 

D Tech 8 D LC50 >10000 ppm N.A. Acceptable Practically 

Non-toxic 

00077316 Bobwhite 

quail 

Colinus 

virginianus 

850.2200

71-2a 

D Tech 8 D LC50 4042 ppm 2150 Acceptable Slightly 

Toxic 

Freshwater Invertebrate Acute Toxicity

00063851 Water flea Daphnia 

magna 

850.1010

72-2 

S 98.0 48 hr EC50 2.2 ppm 0.75 Acceptable Moderately 

Toxic 

70890 

00026469 

Water flea Daphnia 

magna 

850.1010

72-2 

S 10 48 hr EC50 3.1 ppm 1.8 Supplemental Moderately 

toxic 

Freshwater Fish Acute Toxicity

00063850 Bluegill 

sunfish 

Lepomis 

macrochirus 

850.1075

72-1 

S 98 96 hr LC50 4.1 ppm 3.2 Acceptable Moderately 

Toxic 

00063850 Rainbow 

trout 

Oncorhynchus 

mykiss 

850.1075

72-1 

S 98.0 96 hr LC50 1.75 ppm 1.4 Acceptable Moderately 

Toxic 

Estuarine/Marine Fish Acute Toxicity

 

3

http://iaspub.epa.gov/tmdl_waters10/attains_nation.tmdl_pollutant_detail?p_pollutant_group_id=885&p_pollutant_

group_name=PESTICIDES

4

 ACRrainbow trout = Chronic NOAEC/96-hr LC50 = 1.75 ppm/0.75 ppm = 2.33 5

 ACRDaphnia magna = Chronic NOAEC/96-hr LC50 = 2.2 ppm/1.4 ppm = 1.57 
 Page 8 of 12

MRID 

NOs. 

COMMON 

NAME 

TAXONOMIC 

NAME 

GUIDE

LINE TYPE % a.i. 

STUDY 

TIME

DOSE

TYPE

TOXI

CITY 

TOX 

LEVEL NOEL STUDY STATUS 

TOX 

CATEGORY/

41885301 Sheepshead 

minnow 

Cyprinodon 

variegatus 

850.1075

72-3 

F 99.85 96 hr LC50 8.3 ppm 6.7 Acceptable Moderately 

Toxic 

00150032 Sheepshead 

minnow 

Cyprinodon 

variegatus 

850.1075

72-3 

S 98.0 96 hr LC50 17 ppm 7.5 Acceptable Moderately 

Toxic 

Freshwater Fish Early-Life Stage Toxicity

41986701 Rainbow 

trout 

Oncorhynchus 

mykiss 

850.1400

72-4a 

S 99.85 21 D LOEC 1.0 ppm 0.75 Acceptable N/A for 

chronic 

Freshwater Invertebrate Chronic Toxicity 

41885304 Water flea Daphnia 

magna 

850.1300

72-4b 

F 99.85 21 D LOEC 2.6 ppm 1.4 Acceptable Percent 

survival 

Estuarine/Marine Acute Toxicity – Invertebrates, Crustaceans

41885303 Opossum 

shrimp 

Americamysis 

bahia 

850.1035

72-3 

F 99.85 96 hr LC50 2.7 ppm 1.5 Acceptable Moderately 

Toxic 

00150034/ 

46006403 

Daggerblade 

grass shrimp 

Palaemonetes 

pugio 

850.1045

72-3 

S 98.0 96 hr LC50 45 ppm <10 Acceptable Moderately 

Toxic 

Estuarine/Marine Acute Toxicity – Invertebrates, Mollusks

41885302 Eastern 

oyster 

(embryolarvae) 

Crassostrea 

virginica 

850.1055

72-3b 

S 99.85 48hr EC50 2.67 ppm 1.4 Acceptable Moderately 

Toxic 

Mammalian Acute Oral Toxicity

41885501 Rat Rattus 

norvegicus 

870.1100

81-1 

O 99.9 14 D LD50 770(M) 

515(F)

mg/kgbw ------ Acceptable Slightly Toxic

III 

Test Type—O: oral; D: dietary; F: flow-through; S: Static; 

Tox Level—mg/kg-bw: milligrams per kilogram-body weight; ppm: parts per million (equivalent to milligrams per kilogram-diet (mg/kg-diet)

for dietary exposure and milligrams per liter (mg/L) for aquatic exposures) 

Categories of acute toxicity for aquatic organisms (U.S. EPA, 2004) based on EC50/LC50 (mg/L): < 0. 1 very highly toxic; 0.1-1 highly toxic; >1-

10 moderately toxic; >10-100 slightly toxic; >100 practically nontoxic. 

Categories of acute toxicity to terrestrial animals, avian and mammalian (U.S. EPA, 2004). LC50 (ppm): < 50 very highly toxic; 50 - 500 highly 

toxic; 501 – 1000 moderately toxic; 1001-5000 slightly toxic; >5000 practically non-toxic. LD50 (mg/kg bw): < 10 very highly toxic; 10 - 50 

highly toxic; 51 - 500 moderately toxic; 501-2000 slightly toxic; >2000 practically non-toxic.

Adverse Ecological Incidents 

There are no reported incidents in the Agency's Ecological Incident Information System 

(EIIS) regarding DBDCB. The absence of documented incidents does not necessarily mean that 

such incidents did not occur. 

Ecological Effects Risk Assessment 

An ecological risk assessment was not conducted on the DBDCB’s use patterns described 

in this document. The Agency needs to conduct an environmental fate and ecological risk 

assessment for material preservatives discharges and industrial process and water system uses 

and consumer product uses (e.g., dish washing liquids) that may discharge directly into terrestrial 

and aquatic environments or which may reach those environments after passing through waste 

water treatment plants (WWTPs). For this ecological risk assessment, including an endangered 

species assessment, the Agency anticipates needing the ecotoxicity data outlined on page 9 of 

this document. 
 Page 9 of 12

The Agency has not conducted a risk assessment for DBDCB that supports a complete 

endangered species effects determination. The ecological risk assessment planned during 

registration review will allow the Agency to determine whether DBDCB’s use has “no effect or 

“may affect” federally listed threatened or endangered species (listed species) or their designed 

critical habitats. When an assessment concludes that a pesticide’s use “may affect” a listed 

species or its designated critical habitat, the Agency will consult with the U.S. Fish and Wildlife 

Service and/or National Marine Fisheries Services (the Services), as appropriate.

Environmental Fate and Ecotoxicity Data Gaps and Anticipated Work 

• The Agency anticipates needing the following data to conduct a complete environmental 

fate assessment for the DBDCB degradate MGN because it is the primary and terminal 

degradate in many of the environmental fate studies: 

o Activated Sludge Respiration Inhibition (OCSPP 850.6800 or OECD 209); 

o One of four activated sludge biodegradability studies (Ready biodegradability 

[OCSPP 835.3110], or Porous Pot Test [OCSPP 835.3220], or Simulation Tests to 

Assess the Biodegradability of Chemicals Discharged to Wastewater [OCSPP 

835.3280], or Simulation Test – Aerobic Sewage Treatment: A. Activated Sludge 

Units [OCSPP 835.3240]6

: 

• The Agency anticipates needing the following ecotoxicity data in order to conduct a 

complete ecological risk assessment, including an endangered species assessment: 

o Algal toxicity (Tier II) using freshwater green alga, Selenastrum capricornutum 

(OCSPP 850.5400); 

o Algal toxicity (Tier II) using freshwater diatom, Navicula pelliculosa (OCSPP 

850.5400) (If the IC50 is > 1000 ppb a.i. for Selenastrum capricornutum this data can 

be waived; 

o Algal toxicity (Tier II) using marine diatom, Skeletonema costatum (OCSPP 

850.5400) (If the IC50 is > 1000 ppb a.i. for Selenastrum capricornutum this data can 

be waived; 

o Algal toxicity (Tier II) using cyanobacterium, Anabaena flos-aquae (OCSPP 

850.5400) (If the IC50 is > 1000 ppb a.i. for Selenastrum capricornutum this data can 

be waived; 

o Aquatic vascular plant toxicity (Tier II) using Lemna spp. (OCSPP 850.4400) (If the 

IC50 is > 1000 ppb a.i. for Selenastrum capricornutum this data can be waived).

 

6

 The results of the Activated Sludge Inhibition Test (ASRI) determine which of these biodegradation tests are 

required. If the ASRI test EC50 is less than or equal to 20 mg/L, then either the (i) Biodegradation in Activated 

Sludge Study or (ii) Simulation Test – Aerobic Sewage Treatment: A. Activated Sludge Units or the (iii) Porous Pot 

Test is required. If the ASRI test EC50 is greater than 20 mg/L, then the applicant must choose either: (i) Ready 

Biodegradability or (ii) Biodegradation in Activated Sludge or (iii) Simulation Test – Aerobic Sewage Treatment: A. 

Activated Sludge Units or (iv) the Porous Pot Test. If the antimicrobial passes the Ready Biodegradability study, 

then no further testing is required. If, however, the antimicrobial fails the Ready Biodegradability study, then the (i) 

Biodegradation in Activated Sludge or (ii) Simulation Test – Aerobic Sewage Treatment: A. Activated Sludge Units, 

or (iii) the Porous Pot study is required. 
 Page 10 of 12

Appendix A. Environmental Fate Guideline Study Justifications 

Study Title: Activated Sludge, Respiration Inhibition Test for Sparingly Soluble Chemicals 

or Activated Sludge Respiration Inhibition Test 

Guideline Number: OCSPP 850.6800 or OECD 209, respectively 

Test Substance: 2-methylene glutaronitrile (MGN)

Rationale for Requiring the Data

An activated sludge, respiration inhibition test (ASRI) is needed to identify whether MGN, which is 

expected to reach waste treatment facilities based on the use pattern, could harm activated sludge 

microorganisms found in biological wastewater treatment systems. The Agency will use these data to 

determine the potential impact of this degradate on waste water treatment plants (WWTPs) as a result of 

respiration inhibition of activated sludge microorganisms. This test is also necessary to enable the EPA 

to establish correct concentrations for use in the biodegradability test, if that test is determined to be 

adequate for assessing biodegradation potential. Furthermore, the results of the ASRI determine which 

biodegradation test is required. If the ASRI test EC50 is less than or equal to 20 mg/L, then either the 

Biodegradation in Activated Sludge Study or the Porous Pot Test is required. If the ASRI test EC50 is 

greater than 20 mg/L, then the applicant must choose either: (i) Ready Biodegradability or (ii) 

Biodegradation in Activated Sludge or the Porous Pot Test. If the antimicrobial passes the ready 

biodegradability study, then no further testing is required. If, however, the antimicrobial fails the ready 

biodegradability study, then the Biodegradation in Activated Sludge or the Porous Pot study is required.

Practical Utility of the Data

1. How will the data be used?

The results of this study will identify the inhibitory effect of MGN on treatment plant microorganisms’ 

respiration at various concentrations as well as the EC50, the effective concentration at which the respiratory 

rate is reduced by 50%. EPA will use the data to determine the potential for MGN to adversely impact 

activated sludge microorganisms present in a WWTP which, in the event that microorganisms are adversely 

impacted, could result in the subsequent release of less than fully treated or marginally treated wastewaters. 

Additionally, eliciting or not eliciting such releases are important considerations in characterizing impacts 

to the quality of the aquatic environment and subsequent effects to federally listed endangered and 

threatened species and their critical habitat and to non-target non-listed species and their habitat. The data 

will also determine suitable non-inhibitory concentrations to be used in biodegradability tests. 

2. How could the data impact the Agency’s future decision-making?

If concentrations of MGN reaching the influent of the WWTP are expected to be below the level of concern 

identified by the ASRI test, then the Agency can conclude that there is no concern for adverse impacts to 

activated sludge microorganisms, or for release of less than fully treated or marginally treated wastewaters 

that would result from such impacts. If the data indicate a high degree of activated sludge respiration 

inhibition for MGN, a more rigorous test for biodegradation in wastewater treatment will be required than 

the ready biodegradability test. Additionally, if these data indicate that MGN can be inhibitory to activated 

sludge microorganisms at concentrations that may be possible in WWTP influent from antimicrobial uses, 

this may result in the Agency requiring label or use restrictions that would be necessary to meet the “no 

unreasonable adverse effects” requirement of FIFRA. 
 Page 11 of 12

 Study Titles: Ready Biodegradability, Porous Pot Test, Simulation Tests to Assess the 

Biodegradability of Chemicals Discharged to Wastewater (particularly Biodegradation in Activated 

Sludge), or Simulation Test – Aerobic Sewage Treatment: A. Activated Sludge Units, respectively 

Guideline Numbers: OCSPP 835.3110, 835.3220, 835.3280, or 835.3240, respectively 

Test Substance: 2-methylene glutaronitrile (MGN)

Rationale for Requiring the Data

MGN is a significant abiotic and biotic degradate of DBDCB and waste water treatment process 

systems are expected to be exposed to this degradate based on use patterns of DBDCB and known fate 

information. As part of its screening-level environmental fate assessment, EPA will analyze the potential 

impact of the use of DBDCB on the microorganisms in the biological treatment processes of a WWTP. For 

DBDCB, the residue of interest and concern for this analysis is MGN, the twice-debrominated parent 

compound. Results from an activated sewage sludge study are expected to supply information on the rate 

of biological breakdown and the completeness of the degradation to carbon dioxide and water. One of four 

studies can be used to supply the information needed. The results of the Activated Sludge Respiration 

Inhibition Test (ASRI) determine which of these four biodegradation tests are required. If the ASRI test 

EC50 is less than or equal to 20 mg/L, then either the (i) Biodegradation in Activated Sludge Study or (ii) 

Simulation Test – Aerobic Sewage Treatment: A. Activated Sludge Units or the (iii) Porous Pot Test is 

required. If the ASRI test EC50 is greater than 20 mg/L, then the applicant must choose either: (i) Ready 

Biodegradability or (ii) Biodegradation in Activated Sludge or (iii) Simulation Test – Aerobic Sewage 

Treatment: A. Activated Sludge Units or (iv) the Porous Pot Test. If the antimicrobial passes the Ready 

Biodegradability study, then no further testing is required. A ready biodegradability study would allow the 

Agency to determine whether or not the chemical achieves ‘‘pass levels’’ for ready biodegradability (e.g., 

70% removal of dissolved organic carbon). If, however, the antimicrobial fails the Ready Biodegradability 

study, then the (i) Biodegradation in Activated Sludge or (ii) Simulation Test – Aerobic Sewage Treatment: 

A. Activated Sludge Units, or (iii) the Porous Pot study is required. 

Practical Utility of the Data

1. How will the data be used? 

The data from a biodegradation study with MGN will be used to determine the percent removal of this 

degradate as a result of biodegradation by activated sludge microorganisms. Percent removal via 

biodegradation is a key input used to determine concentrations of MGN in surface water immediately 

downstream of wastewater treatment plants. The concentrations of antimicrobial pesticide and degradates 

immediately downstream of wastewater treatment plants are used to assess exposure and risk to non-target 

endangered and non-endangered species. Exposure data are integral parts of the risk equation for 

determining the impact of pesticides to beneficial non-target organisms through direct exposure from their 

food sources and habitat. 

In addition, the data will be used to determine whether or not additional studies and/or label restrictions 

are needed. If the chemical does not achieve a ‘‘pass level’’ for ready biodegradability, then the Agency 

would determine that the chemical has the potential to pass through the WWTP and therefore may be 

discharged in to the aquatic environment potentially resulting in exposure to non-target species. This 

determination would trigger further data requirements to assess the chemical’s impact on the aquatic 

environment upon release. 

2. How could the data impact the Agency’s future decision-making? 

If the chemical is found to be readily biodegraded, then activated sludge microorganisms would be 

identified as highly biodegradable (90% removal via biodegradation). This result would be used in 

conjunction with wastewater treatment plant influent volume and measurement endpoints for aquatic 

organisms to determine concentrations of antimicrobial pesticide downstream of wastewater treatment 

plants. If high inhibition of activated sludge microorganisms (i.e., EC50 less than or equal to 20 mg/L) is 

determined in an activated sludge respiration inhibition test, then higher-tiered wastewater treatment plant 
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(WWTP) biodegradation studies and/or an activated sludge sorption isotherm study may be triggered. If 

results of higher-tier WWTP biodegradation tests indicate low or negligible removal by biodegradation, the 

risk assessments for this chemical would reflect the magnitude of potential risk derived from the chemical’s 

lack of biodegradability. The risk assessment may result in the Agency requiring label restrictions that 

would be necessary for the products to meet the “no unreasonable adverse effects” requirements of FIFRA 

(e.g., changes to the directions for disposal). 

In addition, EPA is required by section 7(a)(2) of the ESA to ensure that any action it authorizes or takes 

“...is not likely to jeopardize the continued existence of any endangered or threatened species or result in 

the destruction or adverse modification of critical habitat” and to use the best scientific data available” in 

carrying out this obligation. The data EPA intends to call in are necessary to inform the determination 

required by the ESA as to whether continued registration of a pesticide is not likely to jeopardize the 

species or its critical habitat. Absent this data EPA may not be able to conclude that this pesticide will not 

harm endangered species and may therefore need to consider other limitations on the use of the pesticide in 

order to fulfill the Agency’s statutory obligations under the ESA. The lack of these data will limit the 

flexibility that the Agency and registrants have in coming into compliance with the ESA and could result in 

use restrictions which are unnecessarily severe.