Toxicity, Sublethal Effects, and Potential Modes of Action 

of Select Fungicides on Freshwater Fish and Invertebrates

By Adria A. Elskus

Open-File Report 2012–1213

U.S. Department of the Interior

U.S. Geological Survey
U.S. Department of the Interior

KEN SALAZAR, Secretary

U.S. Geological Survey

Marcia K. McNutt, Director

U.S. Geological Survey, Reston, Virginia: 2012

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Suggested citation:

Elskus, A.A., 2012, Toxicity, sublethal effects, and potential modes of action of select fungicides on freshwater fish 

and invertebrates: U.S. Geological Survey Open-File Report 2012–1213, 42 p., at 

http://pubs.usgs.gov/of/2012/1213/.

Any use of trade, firm, or product names is for descriptive purposes only and does not imply 

endorsement by the U.S. Government.

Although this information product , for the most part, is in the public domain, it also may contain copyrighted 

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iii

0BAcknowledgments

Critical reviews by Michael Focazio, Christopher Ingersoll, Kathryn Kuivila, and Kelly 

Smalling of the U.S. Geological Survey greatly improved the manuscript. Special thanks are extended to 

Lebelle Hicks, Maine Board of Pesticide Control, and to Emily Monosson for their exacting input on the 

focus, organization, and structure of this review. I thank Christy Finlayson for compiling the initial data 

for this report. Funding was provided by the Contaminants Biology Program and the Toxic Substances 

Hydrology Program of the U.S. Geological Survey.
iv

Contents

8TAcknowledgments8T..........................................................................................................................................................iii

8TAbstract8T ......................................................................................................................................................................... 1

8TIntroduction8T.................................................................................................................................................................... 1

8TPurpose and Scope8T.................................................................................................................................................... 2

8TOverview of Fungicides8T ................................................................................................................................................. 2

8TFungicide Resistance Action Committee8T.................................................................................................................... 2

8TFungicide Modes of Action8T......................................................................................................................................... 3

8TAre Modes of Fungicide Action Related to Biochemical and Toxicological Effects in Fish and Invertebrates?8T .......... 3

8TWhich Organisms, Life Stages, and Endpoints Are Most Sensitive?8T ....................................................................... 12

8TOrganisms and Life Stages8T .................................................................................................................................. 12

8TEcosystem Effects8T................................................................................................................................................ 13

8TImmune Function8T.................................................................................................................................................. 13

8TOxidative Stress8T ................................................................................................................................................... 13

8TEndocrine Effects8T ................................................................................................................................................. 16

8TMixtures8T................................................................................................................................................................ 16

8TDetailed Summaries for Select Fungicides by Fungicide Resistance Action Committee Mode of Action8T .................... 16

8TFungicide Resistance Action Committee Mode of Action B: Mitosis and Cell Division8T............................................. 16

8TZoxamide8T.............................................................................................................................................................. 16

8TEnvironmental Fate in Aquatic Systems8T ........................................................................................................... 16

8TMode of Toxic Action in Fungi8T........................................................................................................................... 17

8TBiochemical Effects in Mammals8T ...................................................................................................................... 17

8TToxic Effects in Freshwater Organisms8T ............................................................................................................ 17

8TRelationship Between Zoxamide Fungal MOA and Effects in Nonfungal Organisms8T ....................................... 17

8TFungicide Resistance Action Committee Mode of Action C: Respiration8T ................................................................. 17

8TBoscalid8T................................................................................................................................................................ 17

8TEnvironmental Fate in Aquatic Systems8T ........................................................................................................... 17

8TMode of Toxic Action in Fungi8T........................................................................................................................... 19

8TBiochemical Effects in Mammals8T ...................................................................................................................... 19

8TToxic Effects in Freshwater Organisms8T ............................................................................................................ 19

8TData Gaps8T......................................................................................................................................................... 19

8TRelationship Between Boscalid Fungal MOA and Effects in Nonfungal Organisms8T.......................................... 19

8TAzoxystrobin8T......................................................................................................................................................... 21

8TEnvironmental Fate in Aquatic Systems8T ........................................................................................................... 21

8TMode of Toxic Action in Fungi8T........................................................................................................................... 21

8TBiochemical Effects in Mammals8T ...................................................................................................................... 21

8TToxic Effects in Freshwater Organisms8T ............................................................................................................ 21

8TData Gaps8T......................................................................................................................................................... 22

8TRelationship Between Azoxystrobin Fungal MOA and Effects in Nonfungal Organisms8T................................... 22

8TPyraclostrobin8T....................................................................................................................................................... 23

8TEnvironmental Fate in Aquatic Systems8T ........................................................................................................... 23

8TMode of Toxic Action in Fungi8T........................................................................................................................... 23

8TBiochemical Effects in Mammals8T ...................................................................................................................... 23

8TToxic Effects in Freshwater Organisms8T ............................................................................................................ 23

8TData Gaps8T......................................................................................................................................................... 24
v

8TRelationship Between Fungicide MOA and Effects in Nonfungal Organisms8T ................................................... 24

8TFungicide Resistance Action Committee Mode of Action D. Amino Acids and Protein Synthesis8T............................ 24

8TPyrimethanil8T.......................................................................................................................................................... 24

8TEnvironmental Fate in Aquatic Systems8T ........................................................................................................... 24

8TMode of Toxic Action in Fungi8T........................................................................................................................... 24

8TBiochemical Effects in Mammals8T ...................................................................................................................... 25

8TToxic Effects in Freshwater Organisms8T ............................................................................................................ 25

8TData Gaps8T......................................................................................................................................................... 25

8TRelationship Between Fungicide MOA and Effects in Nonfungal Organisms8T ................................................... 25

8TFungicide Resistance Action Committee Mode of Action E: Signal Transduction8T.................................................... 25

8TFludioxonil8T ............................................................................................................................................................ 25

8TEnvironmental Fate in Aquatic Systems8T ........................................................................................................... 25

8TMode of Toxic Action in Fungi8T........................................................................................................................... 26

8TBiochemical Effects in Mammals8T ...................................................................................................................... 26

8TToxic Effects in Freshwater Organisms8T ............................................................................................................ 26

8TData Gaps8T......................................................................................................................................................... 26

8TRelationship Between Fungicide MOA and Effects in Nonfungal Organisms8T ................................................... 26

8TFungicide Resistance Action Committee Mode of Action G: Sterol Biosynthesis in Membranes8T ............................. 27

8TMyclobutanil8T ......................................................................................................................................................... 27

8TEnvironmental Fate in Aquatic Systems8T ........................................................................................................... 27

8TMode of Toxic Action in Fungi8T........................................................................................................................... 27

8TBiochemical Effects in Mammals8T ...................................................................................................................... 27

8TToxic Effects in Freshwater Organisms8T ............................................................................................................ 28

8TData Gaps8T......................................................................................................................................................... 28

8TRelationship Between Fungicide MOA and Effects in Nonfungal Organisms8T ................................................... 28

8TFenarimol8T ............................................................................................................................................................. 28

8TEnvironmental Fate in Aquatic Systems8T ........................................................................................................... 28

8TMode of Toxic Action in Fungi8T........................................................................................................................... 28

8TBiochemical Effects in Mammals8T ...................................................................................................................... 28

8TToxic Effects in Freshwater Organisms8T ............................................................................................................ 28

8TRelationship Between Fenarimol Fungal MOA and Effects in Nonfungal Organisms8T ....................................... 29

8TFungicide Resistance Action Committee Mode of Action: Multisite Contact Activity8T ................................................ 29

8TChlorothalonil8T ....................................................................................................................................................... 29

8TEnvironmental Fate in Aquatic Systems8T ........................................................................................................... 29

8TMode of Toxic Action in Fungi8T........................................................................................................................... 30

8TBiochemical Effects in Mammals8T ...................................................................................................................... 30

8TToxic Effects in Freshwater Organisms8T ............................................................................................................ 30

8TRelationship Between Chlorothalonil MOA and Effects in Nonfungal Organisms8T ............................................. 31

8TSummary and Conclusions8T.......................................................................................................................................... 31

8TReferences Cited8T......................................................................................................................................................... 32

8TAppendix 1. Qualitative Toxicity Categories8T.............................................................................................................. 42

Figure

1. Graph depicting the relative toxic potency of a variety of fungicides to Daphnia magna ................................ 14
vi

Tables

1. FRAC mode of action (MOA) and structure for fungicides in this review. ......................................................... 4

2. Fungicide MOA target site in fungi and biochemical effects in mammals and aquatic organisms. ................... 6

3. Physiological effects of select fungicides in mammals and aquatic organisms................................................. 9

4. Lowest reported lethal (EC50, LC50) and NOEC/NOAEC values for aquatic organisms. .............................. 15

5. Physical-chemical properties and bioconcentration factors (BCF) for selected fungicides. ............................ 18

6. Aquatic life benchmarks and toxicity category for select fungicides................................................................ 20

1–1. Qualitative toxicity categories for fish and aquatic invertebrates..................................................................... 42

Abbreviations

a.i. active ingredient

AhR Aryl hydrocarbon receptor

Aldh1a1 aldehyde dehydrogenase

AR Androgen receptor

ATP adenosine triphosphate

b.w. body weight

3Tcgs3T cystathionine gamma-synthase gene

CYP19 cytochrome P450 19 protein (aromatase)

CYP1A cytochrome P450 1A protein

CYP1A1 cytochrome P450 1A1 protein

3TCyp1a13T cytochrome P4501A1 gene

CYP1A2 cytochrome P450 1A2 protein

CYP2B1/2 cytochrome P4502 B1/2 protein

3TCyp2b23T cytochrome P4501B2 gene

CYP2E1 cytochrome P450 2E1 protein

3TCyp3a13T cytochrome P4503A1 gene

CYP3A1/2 cytochrome P450 3A1/2 protein

3TCyp3a23T cytochrome P4503A2 gene

CYP450 cytochrome P450

3TCyp4a103T cytochrome P4504A10 gene

3TCyp513T cytochrome P450 51 gene

CYP51 cytochrome P450 51 protein

cyt b cytochrome b

cyto bc1 cytochrome bc1 (ubiquinol oxidase)

kg kilogram

d day

DMI cytochrome P450-demethylase inhibiting

DNA deoxyribonucleic acid

DT50 length of time needed for 50 percent of the chemical to disappear relative to its initial concentration

DT90 length of time needed for 90 percent of the chemical to disappear relative to its initial concentration

EBDC ethylene bisdithio-carbamate

EBI ergosterol biosynthesis inhibiting

EC50 concentration that effects a response in 50 percent of the organisms

EDC endocrine disrupting compound

EPA U.S. Environmental Protection Agency

ER estrogen receptor

3Terg113T ergosterol biosynthesis gene (lanosterol 14-α-demethylase)

ERα estrogen receptor α

GST glutathione S-transferase

HOG1 a mitogen-activated kinase

IGFB1 insulin-like growth factor 1

Koc organic carbon-water partition coefficient
vii

Kow octanol-water partition coefficient

LC50 concentration that is lethal to 50 percent of the organisms

LOEC lowest observed effects concentration

M molar concentration

MAP mitogen-activated protein

MAP3K7 mitogen-activated protein kinase kinase kinase 7

MAPK1 mitogen-activated protein kinase 1

MOA mode of action

NADPH nicotinamide adenine dinucleotide phosphate (reduced form)

NOAEC no observed adverse effects concentration

NOEC no observed effects concentration

NOEL no observed effects

3Tos-23T osmosensitive-2 gene

3TPcx3T pyruvate carboxylase

3TPpap2b3T phosphatidic acid phosphatase 2B

Qo quinol oxidation site of the mitochrondrial cytochrome bc1 complex

RNA ribonucelic acid

ROS reactive oxygen species

3TSlco1a43T solute carrier family transporter gene

SSD species sensitivity distribution

T4 thyroxine

TNFR tumor necrosis factor receptor

TSH thryoid stimulating hormone

UDPGT uridine diphosphate glycuronyltransferase

3TUdpgtr23T uridine-diphophaste glucuronosyl transferase 2 gene

3TUgt1a13T uridine glucuonosyl transferase 1A1 gene

3TUgt2a13T uridine glucuonosyl transferase 2A1 gene

USGS U.S. Geological Survey
Toxicity, Sublethal Effects, and Potential Modes of Action 

of Select Fungicides on Freshwater Fish and Invertebrates

By Adria A. Elskus

1BAbstract

Despite decades of agricultural and urban use of fungicides and widespread detection of these 

pesticides in surface waters, relatively few data are available on the effects of fungicides on fish and 

invertebrates in the aquatic environment. Nine fungicides are reviewed in this report: azoxystrobin, 

boscalid, chlorothalonil, fludioxonil, myclobutanil, fenarimol, pyraclostrobin, pyrimethanil, and 

zoxamide. These fungicides were identified as emerging chemicals of concern because of their high or 

increasing global use rates, detection frequency in surface waters, or likely persistence in the 

environment. A review of the literature revealed significant sublethal effects of fungicides on fish, 

aquatic invertebrates, and ecosystems, including zooplankton and fish reproduction, fish immune 

function, zooplankton community composition, metabolic enzymes, and ecosystem processes, such as 

leaf decomposition in streams, among other biological effects. Some of these effects can occur at 

fungicide concentrations well below single-species acute lethality values (48- or 96-hour concentration 

that effects a response in 50 percent of the organisms, that is, effective concentration killing 50 percent

of the organisms in 48 or 96 hours) and chronic sublethal values (for example, 21-day no observed 

adverse effects concentration), indicating that single-species toxicity values may dramatically 

underestimate the toxic potency of some fungicides. Fungicide modes of toxic action in fungi can 

sometimes reflect the biochemical and (or) physiological effects of fungicides observed in vertebrates 

and invertebrates; however, far more studies are needed to explore the potential to predict effects in 

nontarget organisms based on specific fungicide modes of toxic action. Fungicides can also have 

additive and (or) synergistic effects when used with other fungicides and insecticides, highlighting the 

need to study pesticide mixtures that occur in surface waters. For fungicides that partition to organic 

matter in sediment and soils, it is particularly important to determine their effects on freshwater mussels 

and other freshwater benthic invertebrates in contact with sediments, as available toxicity studies with 

pelagic species, mainly 1TDaphnia magna1T, may not be representative of these benthic organisms. Finally, 

there is a critical need for studies of the chronic effects of fungicides on reproduction, 

immunocompetence, and ecosystem function; sublethal endpoints with population and community-level 

relevance.

2BIntroduction

With use rates projected to rise dramatically in the next few years (Troy, 2011), fungicides are 

one of the emerging chemical classes of concern in freshwater systems in the United States. Unlike 

herbicides, which have received much attention due to their putative effects at low concentrations with 

vertebrates (for example, atrazine) (Hayes and others, 2002), and unlike insecticides, whose effects on 

nontarget invertebrates and vertebrates have been widely recognized for decades (Carson, 1964; 

Gustafsson and others, 2010), there have been few studies of fungicide biochemical and physiological 
2

effects on nonfungal organisms (Relyea and Hoverman, 2006; Warming and others, 2009). The nine 

fungicides in this review were selected based on their high or increasing use, detection frequency, or 

likely persistence: azoxystrobin, boscalid, chlorothalonil, fludioxonil, myclobutanil, fenarimol, 

pyraclostrobin, pyrimethanil, and zoxamide. Focused surveys conducted by the U.S. Geological Survey 

(USGS) found that these fungicides are transported off-site from a variety of use-setting and into aquatic 

habitats where they may impact sensitive communities. However, data on occurrence remain scarce 

(Gilliom and others, 2006). For the fungicides in this review for which data are available, maximum 

surface-water concentrations have been reported of 4.6 micrograms per liter (μg/L) for azoxystrobin 

(Smalling and Orlando, 2011), 36 μg/L for boscalid (Smalling and Orlando, 2011), 0.433 μg/L for 

chlorothalonil (Scribner and others, 2006; Smalling and Orlando, 2011), 2.6 μg/L for myclobutanil 

(Smalling and Orlando, 2011), and 7.1 μg/L for pyraclostrobin (Smalling and Orlando, 2011).

As for all pesticides, fungicides undergo a registration process overseen by the U.S. 

Environmental Protection Agency (EPA). Pesticide registration is the process through which the EPA

examines the ingredients of a pesticide; the site or crop on which it is to be used; the amount, frequency,

and timing of its use; and storage and disposal practices. The EPA evaluates the pesticide to ensure that 

it will not have unreasonable adverse effects on humans, the environment, and nontarget species (U.S. 

Environmental Protection Agency, 2012b, p. 756).

The data in this review are derived from the EPA and Canadian pesticide registration documents 

and Web sites, the Pesticide Properties Database (University of Hertfordshire), the Fungicide Resistance 

Action Committee Web site, and from primary literature identified through Web of Science, and include 

standard toxicity test species, as well as a variety of nonstandard organisms.

8BPurpose and Scope

Data on the sublethal effects of fungicides in nonfungal organisms, particularly vertebrates and 

invertebrates, are scarce. The purpose of this report is to provide information on the toxic effects to fish 

and aquatic invertebrates of nine fungicides (azoxystrobin, boscalid, chlorothalonil, fludioxonil, 

myclobutanil, fenarimol, pyraclostrobin, pyrimethanil, and zoxamide). Theses fungicides were 

identified as emerging chemicals of concern because of their high or increasing global-use rates, 

detection frequency in surface waters, or likely persistence in the environment. The report provides an

overview of fungicide modes of action; the relationship of these modes of action to acute toxicity in 

nonfungal organisms; and the relative sensitivity of different aquatic species, life stages, and endpoints. 

The bulk of the report consists of summaries for each selected fungicide: (1) physical and chemical 

characteristics as related to environmental fate in aquatic systems; (2) mode of toxic action in fungi; (3)

biochemical and physiological effects in mammals, fish, and aquatic invertebrates (where known); (4) 

the potential of fungal modes of action to predict effects in vertebrates and invertebrates; and (5) data 

gaps related to toxicity testing.

3BOverview of Fungicides

9BFungicide Resistance Action Committee

The development of fungal resistance to fungicides is a continuous and costly problem, leading 

to loss of crops, increased prices for food, and food shortages. The Fungicide Resistance Action 

Committee (FRAC) is a specialist technical group of CropLife International whose purpose is to

“provide fungicide resistance management guidelines to prolong the effectiveness of ‘at 

risk’ fungicides and to limit crop losses should resistance occur” (Fungicide Resistance 

Action Committee, 2012).
3

The FRAC provides a list of fungicides, the FRAC code list, sorted by mode of action and 

resistance risk, which is updated annually to include new and reclassified fungicides.

10BFungicide Modes of Action

Fungicides are classified according to their biochemical mode of action in fungal organisms 

(Fungicide Resistance Action Committee, 2012). Fungicides target basic cellular processes, with many 

inhibiting fungal biosynthesis of sterols or tubulin or cytochrome-c reductase activity (Casida, 2009). 

Most fungicides target single biochemical sites, but a few have multiple targets. The 10 general 

categories are: mitosis and cell division, nucleic acids synthesis, respiration, amino acids and protein 

synthesis, signal transduction, lipids and membrane synthesis, sterol biosynthesis in membranes, glucan 

synthesis, melanin synthesis in cell wall, and host plant defense induction. The 11th mode is multi-site 

contact activity and the 12th classification is for those compounds with fungicidal activity and an 

unknown mode of action. Even for fungicides whose mode of action is known, often only the general 

mechanism has been identified (for example, inhibition of mitochondrial respiration, inhibition of 

ribosomal RNA synthesis, nonsystemic/protectant barriers, and nonspecific enzyme inactivation), while 

the specific target site(s) remain uncertain. As fungi often develop resistance to these toxins, new 

fungicides are continuously being introduced to the environment. Multi-site contact fungicides (for 

example, chlorothalonil) typically remain effective longer than single-site fungicides (Brent and 

Hollomon, 2007); however, single-site fungicides, by attacking specific biochemical targets, may have 

fewer side effects on other biochemical processes or nontarget organisms (Gisi and Sierotzki, 2008).

In contrast to the target organism (fungi), almost nothing is known regarding fungicide toxic 

mechanisms in nontarget organisms. Similarly, while effects on some biochemical pathways have been 

described in mammals and fish, little is known about biochemical pathways affected in invertebrates. 

Using a battery of in vitro, high-throughput screening assays, ToxCast, an EPA program, has 

demonstrated that any given environmental chemical, including fungicides, can perturb numerous 

biochemical pathways in mammals (Judson and others, 2010).

The site-specific fungicides in this review (table 1) include those that inhibit fungal mitosis and 

cell division (zoxamide), respiration (azoxystrobin, pyraclostrobin, boscalid), amino acid and protein 

synthesis (pyrimthanil), signal transduction (fludioxonil), and sterol biosynthesis in membranes 

(myclobutanil, fenarimol); there is one multisite contact fungicide included (chlorothalonil).

1BAre Modes of Fungicide Action Related to Biochemical and Toxicological Effects in Fish and 

Invertebrates?

As many biochemical pathways and processes are conserved across species, modes of fungicide 

action could predict analogous mechanisms of toxicity, target site(s), and (or) toxic effects for nonfungal 

species. A comparison of fungicide modes of action (MOAs) with biochemical and physiological effects 

in vertebrates and invertebrates indicates that fungicides may be targeting the same or related 

biochemical and (or) physiological processes in nonfungal species (tables 2 and 3). Several researchers 

provide evidence supporting this view. Ochoa-Acuna and colleagues (2009) suggest that the adverse 

effects of conazole fungicides in nontarget species may be mediated through cytochrome P450 

pathways common across species. Strong evidence in support of such expectations is provided by 

Mazur and Kenneke (2008) who report similar, and in some cases identical, in vitro metabolite profiles

for conazoles in trout, rat, and human liver. Other examples are provided by chlorothalonil, which exerts 

its toxic effects on fungi by complexing with sulphydryl-containing proteins, leading to depletion of 

glutathione reserves (Arvanites and Boerth, 2001); some of these same thiol-reactive processes are 

affected in fish (Davies, 1985b; Gallagher and others, 1992; Davies and others, 1994) and invertebrates 
4

(Davies and others, 1994; Baier-Anderson and Anderson, 1998, 2000a). Azoxystrobin affects 

respiration in fungi by inhibiting electron transport in mitochondria, leading to cellular oxidative 

stressand disruption of fungal metabolism and growth. Recent studies indicate that azoxystrobin disrupts 

mitochondrial respiration in both fungi (Bartlett and others, 2002; Kim and others, 2007; Gisi and 

Sierotzki, 2008) and fish (Olsvik and others, 2010). Imidazoles, triazoles, and the pyrimidine fungicide 

fenarimol belong to the cytochrome P450-de-methylase inhibiting (DMI) class of fungicides, but disrupt 

other CYP450s, such as aromatase (CYP19) in both mammals and fish, indicating endocrine disruptive 

action is associated with DMI fungicides (Ankley and others, 2005; Hinfray and others, 2006; Sisman 

and Turkez, 2010). While such biochemical insights do not allow cross-species predictions of toxic 

potency, they do provide a first step towards identifying potential MOAs in aquatic invertebrates and 

fish for which mechanistic studies of fungicide action have not been conducted.

Table 1. FRAC mode of action and structure for fungicides in this review.

[MOA, mode of action; FRAC, Fungicide Resistance Action Committee; CAS No., Chemical Abstracts Service registry 

number; see Abbreviations for protein and gene definitions]

FRAC MOA code and

target site Fungicide CAS no. Chemical group Structure

12TB. mitosis and cell

division

12TB3: α-tubulin 

assembly in mitosis

Zoxamide 156052-68-5 Toluamide

12TC. respiration

12TC2: complex II –

succinate 

dehydrogenase

Boscalid 188425-85-6 Pyridine-carboxamide

C3: complex III – cyto 

bc1 (ubiquinol 

oxidase) at Qo site 

(cyt b gene)

Azoxystrobin 131860-33-8 Methoxy-acrylate

C3: complex III – cyto 

bc1 (ubiquinol 

oxidase) at Qo site 

(cyt b gene)

Pyraclostrobin 175013-18-0 Methoxy-carbamate

12TD. amino acids and 

protein synthesis

12TD1: methionine 

biosynthesis 

(proposed)

Pyrimethanil 53112-28-0 Anilino-pyrimidine
5

13TTable 1.13T FRAC mode of action (MOA) and structure for fungicides in this review.—Continued

[MOA, mode of action; FRAC, Fungicide Resistance Action Committee; CAS No., Chemical Abstracts Service registry 

number; see Abbreviations for protein and gene definitions]

FRAC MOA code and

target site Fungicide CAS no. Chemical group Structure

12TE. signal 

transduction12T

E2: MAP/histidinekinase in osmotic 

signal tranduction

Fludioxonil 131341-86-1 Phenylpyrrole H

N

N

O

O

F

F

12TG. sterol biosynthesis 

in membranes

12TG1: C14-

demethylase in 

sterol biosynthesis

(erg11/cyp51)

Myclobutanil 88671-89-0 Triazole N

N

N

N

Cl

G1: C14-demethylase 

in sterol 

biosynthesis 

(erg11/cyp51)

Fenarimol 60168-88-9 Pyrimidine

Cl

HO

N

N

Cl

12TMulti-site contact 

activity

Chlorothalonil 37223-69-1 Chloronitrile

(phthalonitrile)

Cl Cl

Cl

N

Cl

N
6

Table 2. Fungicide MOA target site in fungi and biochemical effects in mammals and aquatic organisms.

[MOA, mode of action; FRAC, Fungicide Resistance Action Committee; NA, no information is available; see Abbreviations for protein and gene definitions]

FRAC MOA

Fungicide

Mitosis and cell divison

Zoxamide

Respiration

Boscalid

Respiration

Azoxystrobin

12TMOA target site

in fungi

Mitotic arrest via binding to β-tubulin, 

inhibiting tubulin polymerization and 

cell division

Complex II: inhibits succinate 

dehydrogenase, blocks ATP production

Complex III:

cytochrome bc1 (ubiquinoloxidase) at Qo 

site (cyt bgene), blocks ATP production

12TBiochemical effects

in mammals

Antimitotic in fungi, but not in mammals 

which completely detoxify it by

metabolizing zoxamide

Increased liver enzymes (alanine 

aminotransferase), gamma-glutamyl 

transferase, induced thryoid adenomas 

but responses considered adapative and 

reversible

Induces CYP1A1

12TBiochemical effects 

in fish

NA NA Alters mitochondrial respiration & transcripts 

for catalase, MAPK1, IGFB1, transferrin, 

TNFR, CYP1A; DNA damage in liver and 

spermatocytes

12TBiochemical effects

in invertebrates

NA NA Possible inihbition of CYP450s

12TReferences Young and Slawecki, 2001; Young and 

others, 2006; Oesch and others, 2010; 

Fungicide Resistance Action

Committee, 2012

Pest Management Regulatory Agency, 2004; 

Toxicology Data Network, 2007; 

Fungicide Resistance Action Committee, 

2012

Bartlett and others, 2002; Cedergreen and 

others, 2006; Kim and others, 2007; 

Rudzok and others, 2009; Bony and others, 

2010; Olsvik and others, 2010; Fungicide 

Resistance Action Committee, 2012
7

13TTable 2. 13TFungicide MOA target site in fungi and biochemical effects in mammals and aquatic organisms.—Continued

[MOA, mode of action; FRAC, Fungicide Resistance Action Committee; NA, no information is available; see Abbreviations for protein and gene definitions]

FRAC MOA

Fungicide

Respiration

Pyraclostrobin

Mitosis and cell divison

Zoxamide

Respiration

Boscalid

12TMOA target site

in fungi

Complex III:

cytochrome bc1 (ubiquinol 

oxidase) at Qo site (cyt b gene), 

produces free radicals

Disrupts methionine biosynthesis (cgs gene) 

inihibiting secretion of membrane-degrading 

enzymes

MAP/histidine-kinase in osmotic 

signaltransduction (os-2, HOG1), alters 

osmoregulation

12TBiochemical effects

in mammals

NA Enhances the hepatic metabolism and excretion of 

thyroid hormone, increases CYP450s and

UDPGT

NA

12TBiochemical effects 

in fish

NA NA NA

12TBiochemical effects

in invertebrates

NA NA NA

12TReferences Bartlett and others, 2002; Pest 

Management Regulatory Agency, 

2003; Fungicide Resistance 

Action Committee, 2012

Milling and Richardson, 1995; Fritz and others, 

1997; Hurley and others, 1998; New York 

State Department of Environmental 

Conservation, 2005; Fungicide Resistance 

Action Committee, 2012

Motoyama and others, 2005; Vetcher and 

others, 2007; Kanetis and others, 2008; 

U.S. Environmental Protection Agency, 

2011c; Fungicide Resistance Action 

Committee, 2012
8

13TTable 2. 13TFungicide MOA target site in fungi and biochemical effects in mammals and aquatic organisms.—Continued

[MOA, mode of action; FRAC, Fungicide Resistance Action Committee; NA, no information is available; see Abbreviations for protein and gene definitions]

FRAC MOA

Fungicide

Respiration

Azoxystrobin

Respiration

Pyraclostrobin

Multi-site contact activity

Chlorothalonil

12TMOA target site

in fungi

C14- demethylase in sterol 

biosynthesis (erg11/cyp51)

C14- demethylase in sterol biosynthesis 

(erg11/cyp51)

Depletes glutathione, inhibits NADPH oxidase 

and glyceraldehyde 3-phosphate 

dehydrogenase (glycolysis)

12TBiochemical effects

in mammals

Perturbed fatty acid, steroid, and 

xenobiotic metabolism 

pathways, binds ERα 

(estrogen receptor), reduced 

retinoic acid, induced 

oxidative stress genes 

Endocrine agonist/antagonist (ER,AR, 

inhibits CYP19 aromatase), alters 

activity of several CYP450 enzymes

Aryl-hydrocarbon receptor (AhR) agonist; 

induced lipid peroxidation; binds 

glutathione

12TBiochemical effects in fish NA Endocrine disruptor (binds AR, inhibits 

CYP19, altered plasma steroids, 

vitellogenin, steroid glucuronidation), 

carbonic anhydrase

Affected immune responses (reduced/increased 

ROS, phagocytic activity, NADPH 

oxidase); altered levels of gluththione, 

GST, thiol, RNA and DNA

12TBiochemical effects

in invertebrates

NA Endocrine disruptive effects, but no 

biochemical mechanism determined

May affect immune function, induced 

glutathione, suppressed ROS production, 

may inhibit the activation of NADPH 

oxidase-like enzyme

12TReferences Okubo and others, 2004; Duft 

and others, 2007; Sun and

others, 2007; Goetz and Dix, 

2009; Hata and others, 2010; 

Fungicide Resistance Action 

Committee, 2012; University 

of Hertfordshire, 2012; 

studies cited in Chen and 

others, 2009

Andersen and others, 2002; Griffiths and 

Howlett, 2002; Isik and others, 2004; 

Thibaut and Porte, 2004; Ankley and 

others, 2005; Janer and others, 2005; 

Hinfray and others, 2006; Serap, 2006; 

Canistro and others, 2008; Hassold and 

Backhaus, 2009; Fungicide Resistance 

Action Committee, 2012

Davies, 1985; Gallagher and others, 1992; 

Davies and others, 1994; Baier-Anderson 

and Anderson, 1998, 2000a, 2000b; U.S. 

Environmental Protection Agency, 1999; 

Long and others, 2003; Suzuki and others, 

2004; Shelley and others, 2009; McMahon 

and others, 2011; Zhao and others, 2011; 

Fungicide Resistance Action Committee, 

2012
9

Table 3. Physiological effects of select fungicides in mammals and aquatic organisms.

[MOA, mode of action; NA, no information is available; FRAC, Fungicide Resistance Action Committee; see Abbreviations for protein and gene definitions; see 

text for references]

FRAC MOA

Fungicide

Mitosis and cell division

Zoxamide

Respiration

Boscalid 

Respiration

Azoxystrobin

12TPhysiological effects 

in mammals

Not mutagenic, mammals detoxify 

mitosis-inhibiting properties

Not genotoxic, neurotoxic, teratogenic or a 

reproductive toxin

Liver and bile duct pathology, not a genotoxic, 

neurotoxic, mutagenic, teratogenic or a 

reproductive toxin

12TPhysiological effects

in fish

NA Lethargy and narcosis Altered biochemical parameters associated with 

mitochondrial respiration, oxidative stress, 

cell proliferation; provoked DNA damage in 

liver and spermatocytes

12TPhysiological effects 

in aquatic 

invertebrates

NA Reduced daphnid fecundity, reduced 

1TChironomid 1Temergence

Altered zooplankton community structure, 

daphnid swimming, fecundity, respiration, 

heart rate; no effect on downstream drift

12TPhysiological effects 

in amphibians

NA NA Little to no effect on survival, fecundity, 

metamorphosis, growth
10

13TTable 3. 13TPhysiological effects of select fungicides in mammals and aquatic organisms.—Continued

[MOA, mode of action; NA, no information is available; FRAC, Fungicide Resistance Action Committee; see Abbreviations for protein and gene definitions; see 

text for references]

FRAC MOA

Fungicide

Respiration

Pyraclostrobin

Amino acid and protein synthesis

Pyrimethanil

Signal transduction

Fludioxonil

12TPhysiological effects 

in mammals

Thymic atrophy and apoptosis of lymph nodes 

under acute, but not chronic, exposure

Enhanced thyroid hormone metabolism and 

excretion, thyroid hyperplasia, hypertrophy, 

decreased T4, increased TSH, increased 

biliary flow, possible carcinogen

Not genotoxic, teratogenic, or 

carcinogenic

12TPhysiological effects 

in fish

NA NA NA

12TPhysiological effects 

in aquatic 

invertebrates

Highly toxic to freshwater mussel glochidia 

and juveniles

NA NA

12TPhysiological effects 

in amphibians

NA NA NA
11

13TTable 3. 13TPhysiological effects of select fungicides in mammals and aquatic organisms.—Continued

[MOA, mode of action; NA, no information is available; FRAC, Fungicide Resistance Action Committee; see Abbreviations for protein and gene definitions; see 

text for references]

FRAC MOA

Fungicide

Sterol biosynthesis in membranes

Myclobutanil

Sterol biosynthesis in membranes

Fenarimol

Signal transduction

Fludioxonil

12TPhysiological effects 

in mammals

Binds estrogen receptor, perturbs steroid, fatty 

acid and xenobiotic metaboism, reduced 

liver retinoic acid

Endocrine disruption Not genotoxic, teratogenic, 

or carcinogenic

12TPhysiological effects 

in fish

Quiescence, loss of equilibrium, surfacing, 

dark coloration

Endocrine disruption, reduced fecundity NA

12TPhysiological effects 

in aquatic 

invertebrates

Induced settling to bottom Endocrine disruptor, imposex in snails, reduced egg 

production, delayed molting, developmental 

deformities

NA

12TPhysiological effects 

in amphibians

NA NA NA
12

12BWhich Organisms, Life Stages, and Endpoints Are Most Sensitive?

As with other chemicals, no particular organism or taxonomic group has been identified as more 

sensitive than another, as stated by Maltby and others (2009):

“...it is not clear which of the three taxonomic groups—vertebrates (fish), invertebrates, primary 

producers—should be the focus of attention for studies with fungicides...semi-field studies with 

fungicides do not suggest one common sensitive group...”.

Sensitivity may be due to (a) the organism and its life stage, (b) ecosystem effects, (c) changes in 

immune function, (d) oxidative stress, and (or) (e) endocrine function. Sensitivity may also be seen in 

the presence of (f) mixtures.

19BOrganisms and Life Stages

Several approaches have been taken to identify the most sensitive species, life stage, and 

endpoints for environmental assessments. Ankley and colleagues (2009) suggest this could be achieved 

based on knowledge of the mechanism(s) of action of a chemical or suite of related chemicals (for 

example, antiandrogens), identification of molecular markers for the affected biochemical pathways, 

and demonstration that alterations in those markers results in significant changes in functional endpoints 

(for example, reproductive success or immune function). This approach combines the EPA’s tiered 

testing framework for aquatic organisms (in vitro tests and short-term in vivo tests) with genomics and 

computational biology to create predictive toxicology tools and is being developed for endocrine 

disrupting chemicals in fish. As noted by these authors, this basic conceptual approach could be used for 

many chemicals and levels of biological organization. With scant information on the effects of 

fungicides on aquatic organisms, developing such an approach for ecosystem level assessment would be 

a formidable challenge.

To determine if there are consistent taxonomic differences in sensitivity to fungicides, and if 

these are related to toxic mode of action, Maltby and others (2009) used numerous datasets for semifield 

and laboratory exposures of aquatic organisms to fungicides to compare the median concentration that 

effects a response in 50 percent of the organisms (EC50) for fungicides with different toxic modes of 

action, to construct species sensitivity distributions (SSDs), and to derive threshold values. These 

authors found fish and invertebrates fell into two broad groups: Fish were less sensitive than 

invertebrates to ethylene bisdithio-carbamate (EBDC) fungicides (note: EBDC fungicides are not 

covered in this review) and to sterol-biosynthesis-inhibiting fungicides, but more sensitive than 

invertebrates to non-EBDC fungicides with multi-site activity. No other significant taxonomic 

differences were found. One conclusion that can be drawn is that there is no one toxic MOA that is 

consistently more toxic to nonfungal organisms than another. A comparison of the relative toxicities of 

fungicides representing all MOAs supports this view (fig. 1).

While cladocerans (1TDaphnia 1Tspp.) are often sensitive to chemical stress, particularly the early 

life stages (Marshall, 1978, cited in Warming and others, 2009), recent work suggests understudied 

groups, specifically freshwater mussels and gastropods, warrant further study. Freshwater mussels can 

be quite sensitive to pesticides, including fungicides (table 4). However, the acute sensitivity of their 

early life stages (glochidia, juveniles) to these chemicals, relative to 1TDaphnia,1T is inconsistent (Bringolf 

and others, 2007b). Our current lack of understanding of hormonal and environmental regulation of 

reproduction in freshwater mussels hinders our ability to study the effects of fungicides on this 

endpoint, which may be even more sensitive than the larval and metamorph stages currently used for 

testing. Freshwater gastropods may be more sensitive to endocrine disruptors than standard EPA test 

organisms (Ducrot and others, 2010). Mattheissen suggests that mollusks, and gastropods in particular, 

should be incorporated as standard test organisms for endocrine disrupting compounds (EDCs) due to 
13

their sensitivity to these chemicals, their ecological importance, and because mollusk species outnumber 

all invertebrate groups, except insects (Matthiessen, 2008).

20BEcosystem Effects

In the few studies of ecosystem effects of fungicides, alterations in the community structure of 

zooplankton and algae are the most frequently noted response; there are few studies on ecosystem 

function. Community changes in fungicide-treated ecosystems have been attributed to both direct 

toxicity and to secondary effects, including interspecific interactions. It has been proposed that declines 

in key grazers, such as daphnids, would decrease grazing, increase algal populations, and decrease water 

transparency, leading to subsequent deterioration of macrophytes (Warming and others, 2009). In this 

light, it becomes clear that single species EC50 values likely do not represent the worst case scenario 

(Gustafsson and others, 2010). There is evidence that fundamental ecosystem processes, such as leaf 

litter breakdown, can be deleteriously affected by fungicide exposure. For example, exposure to 65 μg/L 

tebuconazole for 5 weeks affected fungal biomass and sporulation associated with leaf material and 

affected assimilation efficiency and physiological fitness of freshwater leaf-shredding amphipods, 

pointing to the need for incorporation of fundamental ecosystem processes in aquatic environmental risk 

assessment protocols (Zubrod and others, 2011).

21BImmune Function

Although many fungicides and toxicants provoke biochemical changes consistent with immune 

function, such as altered reactive-oxygen production, this endpoint is seldom studied. The few studies 

found include chlorothalonil effects on reactive oxygen production and macrophage function in isolated 

fish and oyster cells (Baier-Anderson and Anderson, 1998, 2000a, 2000b), and chlorothalonil effects on

innate immunity in whole fish (Shelley and others, 2009).

2BOxidative Stress

In addition to its role in immune response, reactive oxygen is a potent toxin, capable of 

oxidizing most cellular components (for example, nucleic acids, proteins, membranes, and lipids), 

resulting in significant damage, disruption of enzyme activity, and reduction of cellular integrity (Li and 

others, 2010). The brain, with its high density of lipid-rich neural tissue, is particularly susceptible to 

lipid peroxidative damage compared to other organs (Li and others, 2010), indicating oxidative stress 

could lead to behavioral changes. Thus, oxidative stress, as evaluated by reactive oxygen generation, 

lipid damage, and (or) behavioral change, could be an important sublethal endpoint for fungicide 

toxicity studies.
14

Figure 1. Graph depicting the relative toxic potency of a variety of fungicides to 1TDaphnia magna1T grouped by mode of action. (48 hour LC50 

is the concentration that effects a lethal response in 50 percent of the organisms in 48 hours. 1TDaphnia magna1T LC50 values are from 

University of Hertfordshire (2012).
15

Table 4. Lowest reported lethal (EC50, LC50) and NOEC/NOAEC values for aquatic organisms.

[FRAC, Fungicide Resistance Action Committee; MOA, mode of action; 48 h EC50, concentration that effects a response in 50 percent of the organisms by 48 

hours; 96 h LC50, concentration that effects a response in 50 percent of the organisms by 96 hours; NOEC/NOAEC, no-observable effects concentration/no 

observable adverse effects concentration; h, hours; NA, no data available; > , greater than; < , less than; all values are in micrograms per liter]

FRAC MOA

Fungicide

Invertebrate

48 h EC50

Invertebrate

chronic 

NOAECP

a

Fish

96 h 

LC50

Fish

chronic 

NOAECP

a

Freshwater mussels

glochidia, juvenile

EC50

Marine bivalves

96 h shell 

deposition

EC50

Zooplantkon 

community 

effects

Frogs 

Mitosis and cell division

Zoxamide >780 39 156 3.48 NA 715 NA NA

Respiration

Boscalid 5,300P

b 790 2,700 116 NA 1,000 NA NA

Azoxystrobin 260 44 470 147 NA NA <2 10P

c

Pyraclostrobin 15.7 4 6.2 2.35 480 (24 h), 80 (48 h),

30 (96 h)

NA NA NA

Amino acid and protein synthesis

Pyrimethanil 3,000 1,000 10,100 20 NA NA NA NA

Signal transduction

Fludioxonil 900 <19 470 19 NA 370P

d NA NA

Sterol biosynthesis in membranes

Myclobutanil 11,000 NA 2,400 980 NA NA NA NA

Fenarimol 6,800 113P

e 900 180 NA NA NA NA

Multi-site contact activity

Chlorothalonil 54 39 105 8.5 0.97 (48 h), 280 (96 h) 3.6 NA NA

21TP

a

P21TChronic exposure times vary, typically 21 or 28 days.

21TP

b

P21TConsidered an underestimation of toxicity due to problems with test conditions (Aubee and others, 2010b).

21TP

c

P21TChronic exposure to this concentration from fertilization through metamorphosis had no effect (Johansson and others, 2006).

21TP

d

P21TSource: Pest Management Regulatory Agency, 2006b.

21TP

e

P21TNo observable effects concentration.
16

23BEndocrine Effects

Several fungicides in this review have effects consistent with endocrine disruption (tables 2 and

3). However, endocrine endpoints are seldom assessed in fungicide studies, and standard toxicity tests 

may not detect the sublethal effects of endocrine-active chemicals. As used in this review, an endocrine 

disruptor is a chemical that affects hormone action either by altering hormone synthesis and (or)

degradation, or acting as a mimic or antagonist. Endocrine effects can occur at low concentrations, may 

exhibit complex dose-response relationships (for example, inverted U dose-response curves), or be 

manifested in later life stages (Ducrot and others, 2010). New testing guidelines for endocrine disruptors 

are being developed by the Organisation for Economic Cooperation and Development (Organisation for 

Economic Cooperation and Development, 2011). These testing guidelines incorporate partial and full 

life-cycle testing for vertebrates and invertebrates, including aquatic organisms, and should be consulted 

when designing experiments to assess potential endocrine-active fungicides and (or) mixtures. 

Experimental design for testing endocrine-active substances can greatly influence the outcome 

(Matthiessen, 2008). Choice of in vitro and (or) in vivo approaches, cell line, species, life stage, gender, 

length of exposure, endpoints and time points chosen, including generational effects, are among the 

many factors that must be carefully considered.

24BMixtures

Some fungicides only exhibit significant toxicity when combined with other chemicals. Mixtures 

of fungicides, but not individual fungicides, caused endocrine disruption of reproduction in mice 

(Jacobsen and others, 2010). In binary mixtures, azole fungicides increased the toxicity of pyrethroid 

insecticides to daphnids (Norgaard and Cedergreen, 2010; Bjergager and others, 2011). While most 

effects of binary mixtures are fairly well predicted by concentration-addition models, including 

pesticides with different modes of action (Norgaard and Cedergreen, 2010), strong synergism is seen 

with one class of fungicides, the ergosterol biosynthesis inhibiting (EBI) (also known as DMI) 

fungicides. Binary combinations of these pesticides increased insecticide toxicity fourfold to twelvefold 

in 1TDaphnia1T. Pesticides tested to date for synergism include the fungicides prochloraz, epoxiconazole, 

propiconazole, tebuconazole, fenpropidin, fenpropimorph, and azoxystrobin, and the insecticides alphacypermethrin, chlorfenviphos, dimethodate, pirimicarb, and esfenvalerate. Some of these can occur at 

synergizing levels in the environment (175 μg propiconazole/L after a storm event) (Norgaard and 

Cedergreen, 2010). There has been a call for more work on identifying the occurrence of azole 

fungicides in both water and sediment, the effects of pesticide binding to organic matter on pesticide 

toxicity, and additional endpoints and species for evaluating fungicide synergism (Norgaard and 

Cedergreen, 2010).

4BDetailed Summaries for Select Fungicides by Fungicide Resistance Action 

Committee Mode of Action

13BFungicide Resistance Action Committee Mode of Action B: Mitosis and Cell Division

25BZoxamide

34BEnvironmental Fate in Aquatic Systems

Zoxamide may persist in the environment for days to months (U.S. Environmental Protection 

Agency, 2011a), absorbed to sediments and organic matter (table 5). Because it is practically immobile, 
17

it is not expected to leach into groundwater. Once in water, however, it is labile to hydrolysis (DT50 

15.7 days), photolysis (DT50 8 d), and microbial degradation (table 5). Bioconcentration factors for fish 

and invertebrates vary, but are quite low (BCF 115) (table 5) (U.S. Environmental Protection Agency, 

2011a; University of Hertfordshire, 2012).

35BMode of Toxic Action in Fungi

Zoxamide [3,5-dichloro-N-(3-chloro-1-ethyl-1-methyl-2-oxopropyl)-4-methylbenzamide] is a 

benzamide fungicide that acts as an antitubulin, arresting mitosis and cell division (Young and 

Slawecki, 2001; Young and others, 2006; Fungicide Resistance Action Committee, 2012) (table 1).

36BBiochemical Effects in Mammals

Mammals are capable of completely detoxifying the mitosis-inhibiting properties of this 

fungicide, likely through metabolite conjugation with glutathione or glucuronic acid (Oesch and others, 

2010). Zoxamide is not genotoxic and is considered not likely to be a human carcinogen (U.S. 

Environmental Protection Agency, 2001) (tables 2 and 3).

37BToxic Effects in Freshwater Organisms

Zoxamide is categorized by the EPA as highly toxic to freshwater fish and highly to very highly 

toxic to freshwater, marine, and estuarine invertebrates (U.S. Environmental Protection Agency, 2001)

(tables 4 and 6, see appendix 1 for a definition of toxicity categories). The sublethal effects of zoxamide 

on aquatic organisms are unknown.

38BRelationship Between Zoxamide Fungal MOA and Effects in Nonfungal Organisms

The Phase II enzyme pathways mammals use to detoxify zoxamide are also present in aquatic 

vertebrates (Andersson and others, 1985) and active to a limited extent in invertebrates (Navarro and 

others, 2011). However, unlike in mammals, zoxamide is highly toxic to fish and aquatic invertebrates 

(tables 4 and 6). Studies are needed to identify what biochemical pathways are involved in the toxicity 

of this fungicide to aquatic organisms. As zoxamide inhibits cell division in fungi by binding α-tubulin 

and inhibiting-tubulin polymerization, tubulin binding is a likely starting point for investigations of 

zoxamide mechanisms of toxicity in fish and invertebrates

14BFungicide Resistance Action Committee Mode of Action C: Respiration

26BBoscalid

39BEnvironmental Fate in Aquatic Systems

Boscalid is expected to be environmentally persistent (table 5), and while expected to occur in 

low concentrations in surface water and groundwater (Pest Management Regulatory Agency, 2004; 

Aubee and Lieu, 2010a), the USGS has found concentrations as high as 36 μg/L in some surface waters 

(Smalling and Orlando, 2011). Boscalid is quickly depurated in fish (t 16TR

1/2R16T<1 day) (Pest Management 

Regulatory Agency, 2004). Bioaccumulation in benthic invertebrates in contact with sediments has not 

been measured, but may be important due to boscalid partitioning to sediment (Pest Management 

Regulatory Agency, 2004).
18

Table 5. Physical-chemical properties and bioconcentration factors (BCF) for selected fungicides.

[All data are from University of Hertfordshire (2012) except where noted. FRAC, Fungicide Resistance Action Committee; MOA, mode of action; μg/L, 

micrograms per liter; DT50, the length of time needed for 50 percent of the compound to dissipate from the soil (Soil DT50), from water due to hydrolysis

(Hydrolysis DT50), from water due to phololysis (Photolysis DT50); Koc, organic carbon-to-water partition coefficient, that is, the ratio of pesticide 

concentration dissolved in organic carbon and dissolved in water; Kow, the octanol-to-water partition coefficient, that is, the ratio of the pesticide concentration 

in octanol and water; ml/g16TR

ocR16T, milliliters per gram organic carbon; BCF, bioconcentration factor; NA, no data available; NU, data not useful; also see 

Abbreviations]

FRAC MOA

Fungicide

Water 

solubility 

(μg/L)

Hydrolysis 

DT50

(days)

Photolysis 

DT50

(days)

Soil 

DT50 

(days)

Mobility Log 

Kow

Koc

(ml/g16TRocR16T)

Persistence

in water and in soil 

(aerobic)21TP

a

BCF

12TMitosis and cell division

Zoxamide 681 15.7 8 60 Low 3.76 1,124 Low and medium 115

12TRespiration

Boscalid 4,600 Stable 30 200 Low 2.96 1,100 High and high 107

Azoxystrobin 6,700 Stable 8.7 70 Low to medium 2.50 589 Low and medium NA

Pyraclostrobin 1,900 Stable 1.7 32 Immobile 3.99 9304 Low and medium 706

12TAmino acid and protein synthesis

Pyrimethanil 121,000 Stable Stable 55 Medium 2.84 835 Medium and medium NA

12TSignal transduction

Fludioxonil 1,800 Stable 10 164 Immobile 4.12 145-600 Medium and high 366

12TSterol biosynthesis in membranes

Myclobutanil 132,000 Stable 15 560 Low 2.89 NU21TP

b High and high NA

Fenarimol 13,700 Stable 0.5 250 Low to medium 3.69 406-684 High and high 113

12TMulti-site contact activity

Chlorothalonil 810 Stable 65 22 Low 2.94 850 Medium and medium 100

21TP

a

P21TRelative persistence is based on hydrolysis DT50 (water) and Koc (soil) as defined (U.S. Environmental Protection Agency, 2012b #758)

21TP

b

P21TNU-not useful; Koc is not an adequate measure of mobility for myclobutanil as sorption does not appear to be correlated with soil organic carbon, (see U.S. 

Environmental Protection Agency, 2009b, table 2.1)
19

40BMode of Toxic Action in Fungi

Boscalid [2-chloro-N-(4-chlorobiphenyl-2-yl) nicotinamide) is an anilide (carboxamide]

fungicide, disrupting fungal respiration and subsequent ATP production by inhibition of the enzyme 

succinate dehydrogenase via complex II of the mitochondrial electron transport chain (Fungicide 

Resistance Action Committee, 2012) (table 1).

41BBiochemical Effects in Mammals

A range of acute, chronic, developmental, and generational toxicity tests indicates boscalid 

toxicity to mammals is low. In dietary studies with mice exposed for 90 days to 2 years to doses of 2,500 

x 1021TP

3

P21T to 5,000 x 1021TP

3

P21T μg/kg body weight provoked biochemical responses, including increased liver 

enzymes (alanine aminotransferase), increased serum gamma-glutamyl transferase, and induction of 

thyroid follicular cell adenomas; however, the enzyme responses were considered an adaptive response 

to increased metabolic demand, and the thyroid changes were reversible when treatment ceased (Pest 

Management Regulatory Agency, 2004). Boscalid metabolism in mammals is via Phase I and II 

reactions and results in either conjugation with glucuronic acid or sulfate, or the binding of boscalid 

directly to glucuronide with cleavage to sulfate metabolites (Toxicology Data Network, 2007).

42BToxic Effects in Freshwater Organisms

Boscalid is considered moderately to highly toxic to freshwater invertebrates, moderately toxic 

to fish and algae, but highly toxic to marine bivalves (Pest Management Regulatory Agency, 2004; 

Aubee and Lieu, 2010a, 2010b). Boscalid exceeds the EPA’s level of concern for direct, acute risk of 

mortality to listed freshwater fish and aquatic-phase amphibians. Aubee and Lieu stated, “...boscalid 

may result in adverse effects on survival, growth, and (or) fecundity of aquatic animals. There is also 

uncertainty regarding the potential risk to benthic invertebrates, given boscalid’s persistence in water 

and sediment.” (Aubee and Lieu, 2010b, p.13).

43BData Gaps

There are no sublethal studies of boscalid effects on aquatic organisms, and acute and chronic 

tests are missing for some organisms, particularly invertebrates. Recent documents from the EPA 

(Aubee and Lieu, 2010b) indicate that studies of boscalid are needed to evaluate its (1) acute toxicity to 

pelagic freshwater invertebrates (the current boscalid-toxicity value is considered unreliable due to 

problems with precipitates in the test), (2) acute and chronic toxicity to freshwater benthic invertebrates 

using boscalid-spiked sediment, (3) toxicity to freshwater mussels given its high toxicity to oysters, and 

(4) bioaccumulation potential in benthic invertebrates.

4BRelationship Between Boscalid Fungal MOA and Effects in Nonfungal Organisms

Boscalid is basically nontoxic to mammals, but moderately to highly toxic to aquatic organisms 

(table 6). Boscalid may be affecting pathways in aquatic organisms that are different from those reported 

in mammals, or the difference may be explained by toxicokinetics. In mammals, intake via the gut 

allows first-pass metabolism by the liver; intake via the gills in fish does not facilitate this. Biochemical 

effects of boscalid in mammals (for example, alteration of liver enzymes) also do not appear to be 

related to its toxic modes of action in fungi (inhibition of succinate dehydrogenase), and provide no 

insights into potential target sites in aquatic organisms. Thus, with no useful data available on possible 

biochemical targets in aquatic organisms, boscalid’s disruption of energy production in fungi via 

inhibition of the enzyme succinate dehydrogenase provides perhaps the best starting point for 

investigating potential toxic modes of action in fish and invertebrates.
20

Table 6. Aquatic life benchmarks and toxicity category for select fungicides. [FRAC, Fungicide Resistance Action Committee; MOA, mode of action; LC50, concentration that is lethal to 50 percent of 

the organisms; invert, invertebrates]

FRAC MOA

Fungicide

Aquatic life benchmarks21TP

a P21T for fish and 

invertebrates, under acute and chronic exposure, 

in micrograms per liter 

Toxicity category21TP

b

12TMitosis and cell division

Zoxamide 78 and 390 (acute)21TP

c

3 and 39 (chronic)21TP

c

Highly toxic (fish)

Highly to very highly toxic (invert)

12TRespiration

Boscalid 1,350 and NA (acute)21TP

c

116 and 790 (chronic)21TP

c

Moderately to highly toxic (fish)

Moderately to highly toxic (invert)

Azoxystrobin 235 and 130 (acute)

147 and 44 (chronic)

Highly toxic (fish)

Highly toxic (invert)

Pyraclostrobin 3 and 8 (acute)21TP

c

2/4 (chronic)21TP

c

Highly to very highly toxic (fish)

Highly to very highly toxic (invert)

12TAmino acid and protein synthesis

Pyrimethanil 5,000 and 1,520 (acute)21TP

c

1,600 and 970 (chronic)21TP

c

Slightly toxic (fish)

Moderately toxic (invert)

12TSignal transduction

Fludioxonil 115 and 480 (acute)21TP

c

19 and 16 (chronic)21TP

c

Highly toxic (fish)

Highly toxic (invert)

12TSterol biosynthesis in membranes

Myclobutanil 1,200 and 5,500 (acute)

980 and NA (chronic)

Moderately toxic (fish)

Slightly toxic (invert)

Fenarimol 2,050 and 3,400 (acute)

85 and 113 (chronic)

Moderately toxic (fish)

Highly toxic (invert)

12TMulti-site contact activity

Chlorothalonil 5.25 and 1.8 (acute)

3 and 0.6 (chronic)

Very highly toxic (fish)

Very highly toxic (invert)

21TP

a 

P21TThese values are derived from distributions and are threshold criteria below which risks are minimalP P(U.S. Environmental 

Protection Agency, 2012).

21TP

b

P21TU.S. Environmental Protection Agency categories, see Leyhe, 2004, table 1.

21TP

c

P21TEstimated from acute and chronic LC50 values as described (U.S. Environmental Protection Agency, 2012).
21

27BAzoxystrobin

45BEnvironmental Fate in Aquatic Systems

Azoxystrobin is considered to have low-to-medium persistence in the environment (table 5)

(U.S. Environmental Protection Agency, 1997; Bartlett and others, 2002; Pest Management Regulatory 

Agency, 2007). Absorption to sediment, microbial degradation, and indirect photolysis are significant 

pathways of loss, with photodegradation being the most prominent (table 5).

46BMode of Toxic Action in Fungi

Azoxystrobin (methyl (αE)-2-[[6-(2-cyanophenoxy)-4-pyrimidinyl]oxy]-α-

(methoxymethylene)benzeneacetate) is a broad-spectrum systemic fungicide belonging to the group bmethoxyacrylate strobilurins (Fungicide Resistance Action Committee, 2012). In 2002, it was the most 

widely used fungicide in the world (Bartlett and others, 2002) and in 2011, it was the leading product 

driving the fungicide market (Troy, 2011). Strobilurins exert their toxic effects on fungi by inhibiting 

mitochondrial respiration. Specifically, they bind to the quinol oxidation (Qo) site of mitochondrial

cytochrome b, which blocks electron transfer from cytochrome b to cytochrome c1, stopping the 

production of adenosine triphosphate (ATP), and thereby disrupting the energy cycle and preventing 

fungal growth (Bartlett and others, 2002). An additional consequence of this action is the release of 

electrons from the respiratory chain, producing cellular oxidative stress (Kim and others, 2007).

47BBiochemical Effects in Mammals

Azoxystrobin is considered relatively nontoxic to mammals, being rapidly metabolized and 

cleared, mainly via conjugation with glucuronide (Pest Management Regulatory Agency, 2007). The 

main target sites are the bile duct and the liver, resulting in pathogenic changes in these tissues at doses 

of 34,000 μg/kg body weight and above (Pest Management Regulatory Agency, 2007) (tables 2 and 3). 

In the human hepatoma derived cell line HepG2, exposure to 4 x 1021TP

-7

P21T to 2.4 x 1021TP

-4

P21T M (1,600 to 100,850 

μg/L) azoxystrobin produced a dose-related cytochrome P4501A1 (CYP1A1) induction pattern that 

coincided with cytotoxicity (Rudzok and others, 2009). Azoxystrobin is not considered neurotoxic, 

mutagenic, genotoxic, teratogenic, or to be a reproductive toxin. It is classified as an inhalation hazard 

for humans by the European Union (Bartlett and others, 2002).

48BToxic Effects in Freshwater Organisms

In contrast to mammals, azoxystrobin is highly toxic to fish, invertebrates, and freshwater algae

(Pest Management Regulatory Agency, 2007; Ochoa-Acuna and others, 2009) (tables 4 and 6); two of 

its degradates may be slightly toxic to daphnids (U.S. Environmental Protection Agency, 1997). It 

appears to have little adverse effect on amphibians at environmentally relevant concentrations (1 to 10 

μg/L from fertilization to metamorphosis (Johansson and others, 2006).

The effects of azoxystrobin on physiological processes in 1TDaphnia magna1T differ among clones, 

including acute sensitivity (from 71 to 277 μg/L 48-hour EC50s), respiration, age at first reproduction, 

and (increased or decreased) fecundity (Warming and others, 2009). Other physiological endpoints 

affected include swimming velocity, mandible movements, and heart rate, among others (cited in 

Warming and others, 2009; (Friberg-Jensen and others, 2010). Unlike other pesticides shown to increase 

invertebrate drift in lotic systems (Muirhead-Thomson, 1978; Cuffney and others, 1984; Wallace and 

others, 1989; Kreutzweiser and Sibley, 1991; Davies and Cook, 1993; Hose and others, 2002; Beketov 

and Liess, 2008), including the dicarboximide fungicide iprodione (Beketov and Liess, 2008), 
22

azoxystrobin (at 16.5 μg/L NOEC) did not initiate downstream drift in the amphipod 1TGammarus pulex1T

(Beketov and Liess, 2008).

The mechanism by which azoxystrobin exerts its toxic effects in fish is not known; however,

recent work suggests it may, as in fungi, impair mitochondrial respiration. Olsvik and others (2010)

report significant alterations in liver, muscle, and blood parameters associated with mitochondrial

respiration, oxidative stress, and cell growth and proliferation in Atlantic salmon (1TSalmo salar1T) smolts 

exposed to the fungicide formulation Amistar [active ingredient (a.i.) is azoxystrobin] for 4 days, a 

duration representative of their exposure during downstream migration through agricultural runoff. The 

doses used in this study (122 and 352 μg a.i./L) are well above the maximum of 4.6 μg/L measured in 

U.S. surface waters (Smalling and Orlando, 2011). Significant changes in biomarkers of oxidative stress 

(catalase), mitochondrial respiration (MAPK1, IGFBP1,TNFR), stress/biotransformation (CYP1A), and 

general stress (transferrin), as well as plasma glucose and other biochemical parameters, were also noted

in these fish. The authors speculate that long-term exposure could affect fish growth. Studies in 

zebrafish found genotoxic effects (DNA damage) in liver and spermatocytes of adult males exposed to 

an environmentally realistic concentration of azoxystrobin (0.5 μg/L) for 3 weeks (Bony and others, 

2010). Such effects could have population-level impacts by altering xenobiotic metabolism (liver) and 

reproductive success (sperm) (Bony and others, 2010).

In binary mixtures, the toxicity of azoxystrobin to 1TDaphnia magna1T was significantly increased in 

the presence of the imidazole fungicide prochloraz (Cedergreen and others, 2006). The authors suggest 

inhibition of CYP450-related pesticide metabolism as the most likely mechanism.

At low, environmentally relevant concentrations, azoxystrobin may significantly alter ecosystem 

dynamics by deleteriously affecting key species such as daphnids. The most notable effects have been 

seen in freshwater microcosm studies in which 2 μg/L or less azoxystrobin significantly altered 

zooplankton community structure (Gustafsson and others, 2010). Significant effects were also found on 

the composition of microcosm phytoplankton communities, likely due to indirect effects of altered 

zooplankton grazing pressure.

Azoxystrobin appears to have little adverse effect on amphibians at environmentally relevant 

doses. In acute tests, exposure of frog (1TRana temporaria) 1Ttadpoles to azoxystrobin (from 30 to 500 μg/L) 

for 72 hours had no significant effect on survival (Johansson and others, 2006). In the same study, 

chronic exposure to 1 to 10 μg/L azoxystrobin from fertilization through metamorphosis had no effect 

on growth, weight, age at metamorphosis, or survival. Similarly, Belden and others (2010) found direct 

spray application of the 2-fungicide formulation Quilt to toads produced little to no acute toxicity. At the 

Quilt application rate to test chambers (0.13–13 μg/cm21TP

3

P21T propiconazole + 0.076–76 μg/cm21TP

3 P21Tazoxystrobin,

nominal concentrations) yielding nominal concentrations in the water of 7.4–740 μg/L and 4.4–440 

μg/L, respectively, survival of laboratory-reared tadpoles (7 days old) and wild-caught juveniles (~ 60 

days post-metamorphosis) of the toad 1TBufo cognatus 1Twas reduced by 7–10 percent (tadpoles) and 4–22

percent (juveniles) over 72 hours (Belden and others, 2010).

49BData Gaps

Additional information is needed on aquatic metabolism to improve estimates of azoxystrobin 

residues in surface water.

50BRelationship Between Azoxystrobin Fungal MOA and Effects in Nonfungal Organisms

Azoxystrobin affects mitochondrial respiration in fungi, and respiratory-associated changes in 

fish and invertebrates. In both fish and fungi, oxidative stress is also induced. Thus, the effects of 

azoxystrobin in aquatic organisms are consistent with its mechanisms of action in fungi, indicating 

target sites may be similar.
23

28BPyraclostrobin

51BEnvironmental Fate in Aquatic Systems

A major route of dissipation for pyraclostrobin is runoff (U.S. Environmental Protection Agency, 

2011b). Pyraclostrobin is slightly soluble in water, is immobile in soil (though in formulations may be 

slightly to moderately mobile), is minimally photodegraded on soil, but rapidly photodegraded in water 

(Pest Management Regulatory Agency, 2003; European Commission, 2004a) (table 5), with all major 

transformation products extremely short-lived. Indirect photolysis may play an important role in 

degradation, with organic and inorganic constituents of water acting as photosensitizers. Pyraclostrobin

is resistant to hydrolysis (Pest Management Regulatory Agency, 2003). Dissipation rates (DT50) of 

pyraclostrobin in water/sediment systems of 33 days in pond sediments and 9 days in river sediments 

have been reported (European Commission, 2004a). It is considered moderately persistent in sediment.

52BMode of Toxic Action in Fungi

Pyraclostrobin (methyl N-[2-[[[1-(4-chlorophenyl)-1H-pyrazol-3-yl]oxy]methyl]phenyl]-Nmethoxycarbamate) is a synthetic analog of strobilurin A, a natural antifungal metabolite produced by 

the wood-rotting fungus 1TStrobillurus tenacellus1T. Like azoxystrobin, it exerts its toxic effect on fungal 

respiration through inhibition of complex III, cytochrome bc1 (ubiquinol oxidase) at the Qo site (cyt b 

gene) in mitochondria (Bartlett and others, 2002; Fungicide Resistance Action Committee, 2012). In 

addition, the free radicals produced during this process disrupt mitochondrial and cytoplasmic 

membranes, further inhibiting fungal growth (Pest Management Regulatory Agency, 2003).

53BBiochemical Effects in Mammals

No information could be found regarding specific biochemical pathways by which 

pyraclostrobin exerts its toxic effects on mammals, necessitating only a description of general effects.

Pyraclostrobin appears to have only transient effects in mammals. It is rapidly metabolized by rats via 

Phase I and II enzymes, resulting in hydroxylated, demethylated, and glucuronidated metabolites. In 

short-term (from 28 to 90 days) dietary studies, the duodenum was the target organ for all species tested, 

resulting in thickening of the mucosa in mice [30,000 to 40,000 μg/kg body weight/day (bw/d)], 

hyperplasia in dogs (from 9,000 to 9,600 μg/kg bw/d), and ulcers in mice (from 12,900 to 30,400 μg/kg 

bw/d); however some of these effects were not seen with longer exposures, and some fell within 

historical control ranges, making the toxicological significance uncertain. Thymic atrophy and apoptosis 

of the lymph nodes (from 30,000 to 40,000 μg/kg bw/d) in mice, and increased spleen and liver weight 

(from 68,800 to 79,700 μg/kg bw/d) in rats were observed with acute, but not chronic (> 1 year),

exposure. In the absence of life-time studies, it was concluded that pyraclostrobin is not likely 

oncogenic. There was no evidence that it is a reproductive toxin, a teratogen, a neurotoxin, or genotoxic

(Pest Management Regulatory Agency, 2003). It does have developmental effects during embryogenesis 

at maternally toxic doses (European Commission, 2004a).

54BToxic Effects in Freshwater Organisms

Unlike in mammals, pyraclostrobin is highly to very highly toxic to invertebrates, vertebrates, 

and algae in freshwater, estuarine, and marine systems (Pest Management Regulatory Agency, 2003; 

Ochoa-Acuna and others, 2009; U.S. Environmental Protection Agency, 2011b) (tables 4 and 6). 

Pyraclostrobin is more toxic than azoxystrobin, possibly due to its greater lipophilicity (Bartlett and 

others, 2002) (table 5). Studies in bluegill sunfish (1TLepomis macrochirus1T) indicate pyraclostrobin is 

absorbed but rapidly lost, with greater than 90 percent of accumulated residues lost from fish tissues 
24

within 2 to 3 days of depuration (Pest Management Regulatory Agency, 2003; U.S. Environmental 

Protection Agency, 2011b).

Very few studies have reported pesticide effects on freshwater mussels. However, in those that 

have, pyraclostrobin was found to be highly to very highly toxic to the freshwater mussel, 1TLampsilis 

siliquoidea1T, adversely affecting viability of glochidia and reducing juvenile survival (Bringolf and 

others, 2007b). The three fungicides tested (pyraclostrobin, chlorothalonil, and propiconazole) were 

more toxic than any of the other pesticides tested, including fipronil and permethrin (insecticides) and 

atrazine and pendimethalin (herbicides),which were found to not be acutely toxic. Of the three 

fungicides tested, pyraclostrobin (EC50 range 30 to 480 μg/L) and chlorothalonil (EC50 range 40 to 280

μg/L) were over 200 times more toxic than propiconazole (EC50 range 10,000 to 20,750 μg/L). The 

toxicity of sediment-bound fungicides to these sediment-dwelling organisms is unknown but could be 

significant.

5BData Gaps

Whole-sediment acute-toxicity studies for freshwater and marine invertebrates are needed (U.S. 

Environmental Protection Agency, 2011b).

56BRelationship Between Fungicide MOA and Effects in Nonfungal Organisms

Pyraclostrobin is rapidly cleared in mammals via Phase I and II metabolism, which likely 

explains its low toxicity to this group. Although aquatic organisms have Phase I and II metabolic 

capacity, rates of xenobiotic metabolism in these animals are generally much lower than in mammals 

(Stegeman and Hahn, 1994), which may explain the high toxic potency of pyraclostrobin to aquatic 

organisms. However, depuration studies in fish indicate pyraclostrobin is rapidly cleared, with greater 

than 90 percent depurated within the first 2 to 3 days (Pest Management Regulatory Agency, 2003), 

making it unclear why it is so toxic to these aquatic vertebrates. Inhibition of respiration via complex III 

and the disruption of membranes by free radicals in fungi deserve attention as possible target sites of 

pyraclostrobin in aquatic organisms.

15BFungicide Resistance Action Committee Mode of Action D. Amino Acids and Protein Synthesis

29BPyrimethanil

57BEnvironmental Fate in Aquatic Systems

The following information is taken from three reports (New York State Department of 

Environmental Conservation, 2005; Pest Management Regulatory Agency, 2006a; Australian Pesticides 

and Veterinary Medicines Authority, 2010). Pyrimethanil is stable to hydrolysis and photolysis (table 5), 

partitions rapidly from water to sediment, and is expected to persist in anaerobic water/sediment systems

(half-life>365 days) (New York State Department of Environmental Conservation). Aerobic metabolism 

is expected to be the major route of dissipation. Water-sediment DT50 is 80 days (University of 

Hertfordshire, 2012), but accumulation is possible in aquatic systems that are treated repeatedly (U.S. 

Environmental Protection Agency, 2010). Pyrimethanil is not expected to leach into groundwater.

58BMode of Toxic Action in Fungi

Pyrimethanil (4,6-dimethyl-N-phenyl-2-pyrimidinamine) belongs to the anilinopyrimidine 

fungicides (table 1). It disrupts synthesis of the amino acid methionine in fungi, thereby inhibiting 

fungal secretion of enzymes that degrade the cell walls of the host plant that are necessary for fungal 
25

infection (Milling and Richardson, 1995; Fritz and others, 1997; Fungicide Resistance Action 

Committee, 2012).

59BBiochemical Effects in Mammals

Pyrimethanil produces thyroid tumors and exhibits antithyroid activity in rats (Hurley and others, 

1998). Although not all potential sites of action have been studied, pyrimethanil has been reported to 

increase metabolism and excretion of thyroid hormone in the liver via increased hepatic UDPGT activity 

(that metabolizes T4) and serum clearance of T4, increase serum thyroid-stimulating hormone levels, 

and produce cellular hypertrophy, hyperplasia, and (or) increased thyroid weight (specific dose of 

pyrimethanil was not provided, but rather the range of highest dose tested of 21 pesticides, of which 

pyrimethanil was one: from 13,000 to 1,000,000 μg/kg/day, Hurley and others, 1998). All antithyroidal 

effects were reversible following cessation of treatment, consistent with a thyroid-pituitary MOA. The 

EPA classifies pyrimethanil as a possible human carcinogen, the carcinogenic MOA being disruption in 

the thyroid-pituitary status (New York State Department of Environmental Conservation, 2005).

60BToxic Effects in Freshwater Organisms

Pyrimethanil is moderately toxic to invertebrates and slightly toxic to fish (New York State 

Department of Environmental Conservation, 2005; Pest Management Regulatory Agency, 2006a) (tables 

4 and 6).

61BData Gaps

Sublethal studies of pyrimethanil on aquatic organisms have not been conducted. As this 

fungicide rapidly partitions into sediments, effects on potentially sensitive benthic dwellers, such as 

freshwater mussels, should be evaluated.

62BRelationship Between Fungicide MOA and Effects in Nonfungal Organisms

Given the antithyroid action of pyrimethanil in mammals, its effects on the thyroid-pituitary axis, 

thyroid hormone levels, and resulting changes in development and growth should be evaluated in 

aquatic organisms. This is particularly important for amphibians where thyroid hormones play a critical 

role in metamorphosis (Tan and Zoeller, 2007; Laudet, 2011). Given the inhibition of methionine 

biosynthesis in fungi, pyrimethanil effects on synthesis of this amino acid is another potential site of 

action in aquatic organisms.

16BFungicide Resistance Action Committee Mode of Action E: Signal Transduction

30BFludioxonil

63BEnvironmental Fate in Aquatic Systems

Fludioxonil is stabile to hydrolysis but is rapidly photodegraded in water (table 5), with the small 

amount of parent compound that remains (<5 percent) partitioning to sediment where it tends to persist 

(t16TR

1/2 R16T51–154 days) (European Food Safety Authority, 2007). Due to its strong adsorptive properties, it is 

considered immobile in soils; however, its metabolites are highly mobile (European Food Safety 

Authority, 2007). Fludioxonil is not very soluble in water (1,800 μg/L, table 5), and thus is not likely to 

leach into groundwater. It should be noted, however, that because of use patterns (for example, which 

sites, which crops, and rates of application, among other patterns; U.S. Environmental Protection 

Agency, 2012b), the potential for significant runoff is considered high. Although rapidly absorbed 

(fludioxonil levels in whole fish reached 95 percent of the steady state concentration after 13.2 days of 
26

exposure to 10 μg/L), fludioxonil is also rapidly depurated once exposure is terminated (DT16TR

90R16T of 1.8 

days) and does not significantly bioaccumulate in fish (Pest Management Regulatory Agency, 2006b).

64BMode of Toxic Action in Fungi

Fludioxonil [4-(2,2-difluoro-1,3-benzodioxol-4-yl)-1H-pyrrole-3-carbonitrile] is a phenylpyrrole 

fungicide that interferes with the osmoregulation of fungal cells, altering the ability of fungi to sense and 

adapt to osmotic conditions in their environment. Under normal conditions, fungi respond to high 

osmolarity by synthesizing and accumulating glycerol as a compatible solute. Fludioxonil acts as an 

osmotic mimic, inappropriately stimulating glycerol synthesis via the mitogen-activated protein kinase 

pathway, leading to excessive intracellular glycerol accumulation that results in hyphal swelling, germ 

tube abnormalities, and cell lysis (Kanetis and others, 2008). Specifically, fludioxonil disrupts the 

histidine kinases in the Os-1 family involved in osmotic stress signal transduction (Motoyama and 

others, 2005; Vetcher and others, 2007; Fungicide Resistance Action Committee, 2012) and inhibits the 

protein kinase in glycerol biosynthesis (table 1).

65BBiochemical Effects in Mammals

Fludioxonil is not acutely toxic to mammals by oral, dermal, or inhalation routes of exposure, 

does not irritate skin or eyes, and exhibits no genotoxic, teratogenic, or carcinogenic potential (European 

Food Safety Authority, 2007).

6BToxic Effects in Freshwater Organisms

Fludioxonil is highly toxic to aquatic invertebrates and fish under both acute and chronic 

exposures (U.S. Environmental Protection Agency, 2011c) (tables 4 and 6), highly toxic to freshwater 

algae and marine invertebrates (oysters, mysids), and moderately toxic to estuarine fish (sheepshead 

minnows) (Pest Management Regulatory Agency, 2006b). In contrast to the highly toxic parent 

compound, the major fludioxonil transformation products are not acutely toxic to fish or to aquatic 

invertebrates (European Food Safety Authority, 2007).

67BData Gaps

As of December 2011, the EPA indicated that the main data gaps for fludioxonil are its 

ecological effects on aquatic organisms and plants. These include the need for assessment of acute 

toxicity of fludioxonil to 1TDaphnia magna1T and rainbow trout, effects on invertebrate reproduction and 

fish life cycle, and chronic sediment toxicity to invertebrates, 1THyalella azteca1T and 1TChironomus dilutes1T

(U.S. Environmental Protection Agency, 2011d).

68BRelationship Between Fungicide MOA and Effects in Nonfungal Organisms

Nothing is known regarding the sublethal effects of fludioxonil on aquatic organisms, despite its 

high-toxic potency to this group. Although there is no homology known between the osmoregulatory 

pathways of fungi and those of vertebrate and (or) invertebrate groups, the potential for fludioxonil to 

interfere with osmotic regulation in aquatic organisms should be investigated. With no biochemical or 

molecular information on MOAs in nonfungal organisms, it is prudent to begin investigations of 

potential target sites in aquatic organisms by examining fludioxonil effects on mitogen-activated protein 

kinase pathways and histidine kinases, coupled with physiological measurements of osmoregulatory 

function.
27

17BFungicide Resistance Action Committee Mode of Action G: Sterol Biosynthesis in Membranes

31BMyclobutanil

69BEnvironmental Fate in Aquatic Systems

Myclobutanil is environmentally stable to hydrolysis and photolysis, is persistent (water DT50

626 days) (University of Hertfordshire, 2012), and has some mobility, the primary routes of dissipation 

being leaching, runoff, and spray drift (U.S. Environmental Protection Agency, 2009a)(table 5). There is 

some potential for atmospheric transport. Although valid measurements of Koc (organic carbon-water 

partition coefficient, that is, the partitioning of a compound between organic carbon and water) are not 

available, the EPA concludes that the Koc is probably low enough that it would not accumulate in 

sediment (U.S. Environmental Protection Agency, 2009a). Due to the low values for Kow (octanolwater partition coefficient, that is, the partitioning of a compound between octanol and water) for both 

parent and degradation products (mainly the 1,2,4-triazole degradate), it is not expected to 

bioaccumulate (U.S. Environmental Protection Agency, 2009a).

70BMode of Toxic Action in Fungi

Myclobutanil [α-butyl-α-(4-chlorophenyl)1H-1,2,4-triazole-1-propanenitrile] is a DMI triazole

(table 1) that disrupts fungal membranes by inhibiting sterol biosynthesis (University of Hertfordshire, 

2012). Specifically, it acts on the 1Terg111T gene responsible for encoding sterol C14-demethylase

(cytochrome P450 isozyme CYP51), inhibiting demethylation in membrane sterol biosynthesis and,

thereby, preventing synthesis of ergosterol, a major membrane component in fungi (Hata and others, 

2010; Fungicide Resistance Action Committee, 2012). Other DMI fungicide classes include piperazines, 

pyrimidines, pyridines, imidazoles, and triazoles (Hassold and Backhaus, 2009).

71BBiochemical Effects in Mammals

As a group, conazoles have a diversity of toxicological effects in mammals, including cancer 

(propiconazole and triadimefon), altered reproduction (myclobutanil and triadimefon), and altered 

hepatic enzymes (propiconazole, triadimefon, and myclobutanil) (Goetz and Dix, 2009; Chen and 

others, 2009). These effects are likely associated with their ability to induce detoxifying cytochrome 

P450s while inhibiting P450s involved in steroid and steroid hormone biosynthesis (Chen and others, 

2009), including CYP51, an enzyme required for sterol biosynthesis in eukaryotes (Goetz and Dix, 

2009). Some of the biochemical and physiological effects reported in mammals for myclobutanil are 

summarized in tables 2 and 3.

In in vitro assays using human breast cancer MCF-7 cells, myclobutanil competitively bound 

estrogen receptor, suggesting myclobutanil may have antiestrogenic activity (Okubo and others, 2004). 

From genomic studies in male rats, it was inferred that myclobutanil and other triazole fungicides

(propiconazole and triadimefon) perturb steroid, fatty acid, and xenobiotic metabolism pathways by 

altering the expression of genes involved in phase I, II, and III metabolism (1TAldh1a11T, 1TCyp1a11T, 1TCyp2b21T, 

1TCyp3a11T, 1TCyp3a21T, 1TSlco1a41T, and 1TUdpgtr21T), fatty acid metabolism (1TCyp4a101T, 1TPcx1T, 1TPpap2b1T), and steroid 

metabolism (1TUgt1a11T, 1TUgt2a11T) (Goetz and Dix, 2009). In male mice receiving four daily intraperitoneal 

injections (270,000 μg/kg/d), myclobutanil reduced liver levels of retinoic acid (Chen and others, 2009), 

a chemical that inhibits the proliferation of epithelial cells, including breast cancer cells (Chen and 

others, 2009). Rats dosed daily by gavage with myclobutanil (75,000 and 150,000 μg/kg/d) for 14 days 

exhibited significantly increased levels of hepatic mRNA for 1TCyp2b11T, 1TCyp3a23/3a11T, and 1TCyp3a21T, and 

induced activities of the cytochrome P450 enzymes pentoxyresorufin o-depentylase and 

benzyloxyresorufin o-debenzylase (Sun and others, 2007).
28

72BToxic Effects in Freshwater Organisms

Myclobutanil is only slightly toxic to invertebrates and moderately toxic to fish (U.S. 

Environmental Protection Agency, 2009b) (tables 4 and 6). Like other conazoles, it is rapidly 

metabolized by fish (t 1⁄2 2.1 days) and is not expected to biomagnify (Konwick and others, 2006).

A recent EPA report and its appendix (U.S. Environmental Protection Agency, 2009a, b) state 

that myclobutanil is likely to adversely affect the California red-legged frog, directly and (or) indirectly, 

by affecting its critical habitat. No direct toxicity data are available for the aquatic life stages of this 

species, and risk decisions are based on toxicity studies on eggs and larvae of fish, and indirect effects 

on prey (aquatic invertebrates). The report concludes that indirect effects on the aquatic phases of the 

frog’s life history, based on reduction in prey base, are not expected.

73BData Gaps

According to the EPA (U.S. Environmental Protection Agency, 2009a), there are no chronic 

exposure data on myclobutanil toxicity to freshwater invertebrates. Thus, qualitative assessments made 

by the EPA are based on similar DMI triazole fungicides, the open literature, and incident data.

74BRelationship Between Fungicide MOA and Effects in Nonfungal Organisms

As a DMI fungicide that affects a multitude of cytochrome P450 enzymes in mammals, and at 

least one (CY51) in fungi, cytochrome P450s involved in steroid hormone and xenobiotic metabolism

are likely sites of myclobutanil action in aquatic organisms. Physiological repercussions of myclobutanil 

regulation of these P450s include effects on organism development, reproduction, and ability to 

metabolize contaminants.

32BFenarimol

75BEnvironmental Fate in Aquatic Systems

Due to its relatively high Kow (table 5), fenarimol partitions rapidly into sediments and is likely 

to be environmentally persistent. Being photolabile, fenarimol is expected to rapidly degrade in

“’shallow, clear, well-lit water bodies’ but to persist in ‘deep, turbid, poorly illuminated water’” (US 

Environmental Protection Agency, 2007a). It has low potential to bioaccumulate in fish (US 

Environmental Protection Agency, 2007a).

76BMode of Toxic Action in Fungi

Like myclobutanil, fenarimol [α-(2-chlorophenyl)-(4-chlorophenyl)-5-pyrimidinemethanol]

belongs to the sterol DMI fungicides, inhibiting C14-demethylase (CYP51), which is involved in the 

synthesis of the essential fungal membrane sterol, ergosterol (Griffiths and Howlett, 2002; Fungicide 

Resistance Action Committee, 2012).

7BBiochemical Effects in Mammals

In in vivo studies with rats, a single intraperitoneal dose of fenarimol (200,000 ug/kg/bw)

affected several cytochrome P450 enzymes, including CYP3A1/2, CYP2E1, CYP2B1, CYP1A1, and 

CYP1A2 (Paolini and others, 1996). In in vitro studies using human cells (MCF-7) and human placental 

microsomes, fenarimol has an array of estrogenic, antiestrogenic, and antiandrogenic effects, including 

inhibition of aromatase (CYP19) (Vinggaard and others, 2000; Andersen and others, 2002) (table 2).

78BToxic Effects in Freshwater Organisms

Fenarimol is moderately to highly toxic to fish and invertebrates (US Environmental Protection 

Agency, 2007a) (tables 4 and 6) with a variety of biochemical and physiological effects reported (tables 
29

2 and 3). The endocrine disruptive effects of fenarimol deleteriously affect reproduction and 

development in both vertebrates and invertebrates (Hassold and Backhaus, 2009). When added to 

testicular microsomes from carp, fenarimol increased synthesis of ovarian maturation-inducing 

hormones and inhibited hormone clearance pathways, including glucuronidation of testosterone and 

estradiol (Thibaut and Porte, 2004). In studies with fathead minnows, a 21-day exposure to fenarimol 

reduced fecundity (569 μg/L), increased spermatogonia in the testes (569 μg/L), increased oocyte atresia 

(569 μg/L), and altered steroid and vitellogenin levels (96 μg/L) (Ankley and others, 2005). Fenarimol 

significantly inhibits the activity of brain and ovarian aromatase (CYP19) in vitro (Ankley and others, 

2005; Hinfray and others, 2006) but has no effect on brain aromatase in vivo (569 μg/L) (Ankley and 

others, 2005). Fenarimol binds the fathead minnow androgen receptor (Ankley and others, 2005). In in 

vitro studies with several fish species, fenarimol reduced carbonic anhydrase activity, a key regulator of 

salt- and acid-base balance in fish (Isik and others, 2004; Dogan, 2006).

Fenarimol affects reproduction and development in aquatic invertebrates. A 5-month exposure to 

fenarimol-induced imposex and reduced fertility or embryo production in the freshwater prosobranch 

snail, 1TMarisa cornuarietis 1T(EC10=0.0186 μg/L) (Duft and others, 2007). In 1TDaphnia1T neonates exposed 

for 21 days, fenarimol reduced fecundity (EC50= 377.6 μg/L), delayed molting (EC50=430 μg/L), and 

increased the percentage of malformed offspring (EC50=400 μg/L), including eye malformations, which 

may be related to inhibition of ecdysteroid synthesis (Hassold and Backhaus, 2009). At a concentration 

of 100 μM (33,120 μg/L), fenarimol did not affect testosterone metabolism in in vitro microsomal 

fractions of whole-animal homogenates of the freshwater snail 1TM. cornuarietis1T or the amphipod, 

1THyalella azteca1T, but did significantly increase formation of testosterone metabolites in homogenates of 

the echinoderm, 1TParacentrotus lividus1T (Janer and others, 2005).

79BRelationship Between Fenarimol Fungal MOA and Effects in Nonfungal Organisms

As for other DMI fungicides, fenarimol inhibition of fungal CYP51 appears predictive of the 

inhibition of a variety of CYP450s in both vertebrates and invertebrates, disrupting reproduction and 

development.

18BFungicide Resistance Action Committee Mode of Action: Multisite Contact Activity

3BChlorothalonil

80BEnvironmental Fate in Aquatic Systems

The main pathway of chlorothalonil dissipation is via microbial degradation, with degradation 

rates faster under wet, flooded, or aquatic conditions (U.S. Environmental Protection Agency, 1999; 

Extension Toxicology Network (EXTOXNET), 2010). Half-lives in water vary from hours to days 

(Szalkowski and Stallard, 1977; Davies, 1988; Ernst and others, 1991; Caux and others, 1996; State of 

California, 1999; U.S. Environmental Protection Agency, 1999; Extension Toxicology Network 

(EXTOXNET), 2010). Concentrations of chlorothalonil and its primary degradate, SDS-3701, are 

expected to be higher in sediment than in water (U.S. Environmental Protection Agency, 1999). 

Leaching of the parent compound to groundwater is likely to be low (Krawchuk and Webster, 1987; 

U.S. Environmental Protection Agency, 1999; Extension Toxicology Network (EXTOXNET), 2010)

(table 5).

The bioaccumulation potential of chlorothalonil is low in fish, but in bivalves, chlorothalonil 

bioconcentration is above the 1000x threshold of concern (U.S. Environmental Protection Agency, 1999, 

2007b), making bioconcentration by freshwater mussels of potential concern. While less 

environmentally persistent than other chlorinated organic compounds (U.S. Environmental Protection 
30

Agency, 1999), chlorothalonil residues are nonetheless often found in freshwater biota (Caux and others, 

1996).

81BMode of Toxic Action in Fungi

Chlorothalonil (2,4,5,6-tetrachloro-1,3-benzenedicarbonitrile) has been in use for decades. Due 

to its multi-site contact-activity MOA (table 1), it is difficult for fungi to develop resistance against 

chlorothalonil, and it remains among the top five products driving the fungicide market (Troy, 2011). 

Chlorothalonil exerts its toxic effects through binding to and depletion of glutathione, a nonenzymatic 

antioxidant critical to the function of several enzymes important in detoxification and cellular respiration

(Zhao and others, 2011; Fungicide Resistance Action Committee, 2012).

82BBiochemical Effects in Mammals

Chlorothalonil is considered ”practically nontoxic” for acute effects to mammals (U.S. 

Environmental Protection Agency, 1999), but a variety of biochemical and physiological effects have 

been reported (tables 2 and 3). In rats, it is a renal toxin (175,000 μg/kg bw/d, from 23 to 29 months) 

(International Agency for Research on Cancer (IARC), 1999). In a study where nursing rats were treated 

with VanoxTM (a.i. chlorothalonil) by intraperitoneal injection (200,00; 400,000; or 800,000 μg a.i./kg

bw), their offspring exhibited developmental effects and delayed sexual maturation at all doses (Lúcia 

Scherholz de Castro and Heloísa Chiorato, 2007). Chlorothalonil activated the aryl hydrocarbon receptor 

(AhR) in human and rat liver cell lines (Long and others, 2003), and induced lipid peroxidation and 

cytotoxicity, likely via CYP450-mediated metabolism, in isolated rat hepatocytes (Suzuki and others, 

2004). Metabolism of chlorothalonil in rats and dogs indicates chlorothalonil binds to glutathione or to 

cysteine-S-conjugates in the liver (U.S. Environmental Protection Agency, 1999). The EPA lists 

chlorothalonil as a probable human carcinogen (U.S. Environmental Protection Agency, 1999).

83BToxic Effects in Freshwater Organisms

Chlorothalonil is considered very highly toxic to fish and to a range of aquatic invertebrate 

species (Davies and White, 1985; U.S. Environmental Protection Agency, 2007b) (tables 4 and 6). In 

contrast, the main degradate (SDS-3701) is only slightly toxic (U.S. Environmental Protection Agency, 

2007b).

In fish, sublethal effects of chlorothalonil include altered hatching success and survivability 

between 3 μg/L (NOEL) and 6.5 μg/L (LOEL) in fathead minnows (U.S. Environmental Protection 

Agency, 2003), increased respiration in fish at 0.3 μg/L (LOEC), and biased sex ratios and reduced 

activity in medaka (1TOryzias latipes1T) fry at 0.06 μg/L (Teather and others, 2005).

There is some information on mechanisms by which chlorothalonil may exert its toxic actions in 

fish and amphibians (table 2). In fish, chlorothalonil exposure lowered hepatic thiol and altered 

glutathione and glutathione S-transferase (GST) levels (Davies, 1985a; Gallagher and others, 1992; 

Davies and others, 1994). Chlorothalonil impaired gill function in rainbow trout (1TSalmo gairdneri1T) at 2 

μg/L by reducing diffusive capacity (Davies, 1987) and altered immune function as evidenced by altered 

production of reactive oxygen species (Baier-Anderson and Anderson, 1998, 2000b; Shelley and others, 

2009). The altered ability to generate reactive oxygen species (ROS) may occur via chlorothalonil 

inhibition of NADPH oxidase by binding to its sulfhydryl groups (Baier-Anderson and Anderson, 

2000b). Recent work on tadpoles demonstrates that chlorothalonil also has significant effects on 

corticosterone levels at doses <16.4 μg/L (McMahon and others, 2011).

The few studies of sublethal effects of chlorothalonil in freshwater invertebrates also report 

effects on thiol-containing biochemicals and ROS. A 10-day exposure to chlorothalonil increased levels 

of whole-body GST (1.8 μg/L) and whole-body glutathione (0.3 μg/L) in the crustacean 1TParatya 
31

australiensis1T (Davies and others, 1994), and suppressed ROS production in oyster hemocytes (100 μg/L, 

in vitro) (Baier-Anderson and Anderson, 2000a). In the latter study, the authors suggest that 

chlorothalonil may have effects similar to those in fish (inhibition of an NAD[P]H oxidase-like enzyme)

and conclude that chlorothalonil is likely to interfere with phagocyte (immune) function in invertebrates.

Bivalves are considered to be at particular risk from chlorothalonil exposure. Water-borne 

chlorothalonil was highly toxic to glochidia (24h EC50=90 μg/L, 48h EC50=40 μg/L) and juvenile life 

stages (96h EC50=280 μg/L) of freshwater mussels 1TLampsilis siliquoidea1T (Bringolf and others, 2007a), 

1TDreissena polymorpha1T (glochidia 48h EC50=0.97 μg/L), and 1TUno elongates1T (48h EC50=1,847 μg/L, 

glochidia) (Faria and others, 2010). Whether chlorothalonil exposure from sediments represents an 

additional risk to freshwater mussels is unknown (Bringolf and others, 2007a). Sublethal effects of 

chlorothalonil on freshwater mussels have not been studied; however, oysters are 10 to 40 times more 

sensitive to chlorothalonil than fish with appreciable ability to bioconcentrate this fungicide (table 5) and 

are considered representative of freshwater mussels in this regard (U.S. Environmental Protection 

Agency, 1999). Based on studies in oyster hemocytes, many, if not all, organic pollutants, including 

fungicides, may be hazardous to bivalve defense systems (Gagnaire and others, 2006).

84BRelationship Between Chlorothalonil MOA and Effects in Nonfungal Organisms

The fungal MOA of chlorothalonil appears to be strongly predictive of its effects on nonfungal 

organisms. In fungi, chlorothalonil exerts its toxic effects through binding to and depleting glutathione, a 

nonenzymatic antioxidant critical to the function of several enzymes important in detoxification, 

reactive oxygen production, and cellular respiration. In mammals, fish, and invertebrates, chlorothalonil 

also affects glutathione and enzymes and processes associated with it.

5BSummary and Conclusions

The fungicides covered in this review represent those detected most frequently and (or) at the 

highest concentrations, with high or increasing use, or have physical and (or) chemical properties 

indicating they may be persistent in surface waters, making them among the most relevant for closer 

examination of effects in nontarget organisms. A review of the literature reveals that fungicide mode of 

toxic action in fungi is sometimes tantalizingly reflective of the biochemical and (or) physiological 

effects observed in vertebrates and invertebrates; however, far more studies are needed to explore the 

potential to predict effects based on specific fungicide modes of toxic action. There are very few studies 

at the ecosystem level, with most examining changes in ecosystem structure and very few examining 

changes in ecosystem function, arguably a more relevant endpoint. This is especially important given 

that single species LC50 values (the acute concentration that effects a response in 50 percent of the 

organisms), used to indicate the lower limit for acute toxicity, appear to dramatically underestimate the 

toxic potency of some fungicides on ecosystem processes. Mixture studies consistently indicate 

fungicides have additive, and in some cases synergistic, effects. Synergistic effects are particularly 

evident with cytochrome P450-demethylase inhibiting fungicides, and studies with additional fungicides 

are needed. Basic acute- and chronic-toxicity data are missing or inadequate for several fungicides, 

including boscalid, a recently introduced fungicide that is being found consistently in surface waters 

across the United States in relatively high concentrations. For fungicides that are particle reactive and 

persistent in sediments, their effects on freshwater mussels and other freshwater benthic invertebrates 

are particularly important to determine, as available toxicity studies with pelagic species, mainly 

1TDaphnia magna1T, may not be representative of these benthic species. Finally, there is a critical need for 

chronic studies of fungicide effects on sublethal endpoints with population- and community-level 

relevance, such as reproduction, immunocompetence, and ecosystem function.
32

The U.S. Environmental Protection Agency pesticide registration process uses existing and new 

data to ensure each pesticide registered will have no “unreasonable adverse effects on humans, the 

environment, and nontarget species” (U.S. Environmental Protection Agency, 2012). However, as 

recognized by the U.S. Environmental Protection Agency registration review process (U.S. 

Environmental Protection Agency, 2012), the state of the sciences in risk assessment, toxicology, and 

environmental chemistry continues to evolve. Consequently, there will continue to be new scientific 

understandings of the active, as well as adjuvant, ingredients in pesticides and their formulations 

regarding environmental fate and transport, as well as potential biological effects. The compilation 

summarized in this paper addresses and reveals data gaps in our scientific understanding of the targeted 

fungicides as potential environmental contaminants. Therefore, information such as this could be useful 

to the U.S. Environmental Protection Agency and other agencies in registration and registration review 

activities, as well as for the larger scientific community engaged in new and ongoing research on the 

potential environmental-health impacts of fungicide use.

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7BAppendix 1. Qualitative Toxicity Categories

Table 1–1. Qualitative toxicity categories for fish and aquatic invertebrates. [LC50, lethal concentration that kills 50 percent of the organisms; EC50, effective 

concentration that immobilizes 50 percent of the organisms; table reprinted from Leyhe, 2004]

LC50 or EC50 (μg/L) Category

<100 Very highly toxic

100–1,000 Highly toxic

>1,000–< 10,000 Moderately toxic

>10,000–< 100,000 Slightly toxic

>100,000 Practically nontoxic