PC Code Numbers: 

110301 (Acid Blue 9) 

110302 (Acid Yellow 23) 

110303 (Aquashade, 23.63% Acid Blue 9 and 2.39% Acid Yellow 23) 

DP Barcode: D302 183 

Date: September 26,2005 

MEMORANDUM 

SUBJECT: 

TO: 

FROM; 

Ecological Risk Assessment of End-Use Products Containing the Dyes Acid Blu2 

9 and Acid Yellow 23 (Aquashade, 23.63% Acid Blue 9 and12.39% Acid Yellow 

23; Aquashade OA, 2.36% Acid Blue 9 and 0.24% Acid Yellow 23; PondCare 

Algae Blocker, 2.36% Acid Blue 9 and 0.24% Acid Yellow 23; Admiral Liquid, 

15.3 1 % Acid Blue 9 and 1.00% Acid Yellow 23; Admiral WSP, 49.72% Acid 

Blue 9 and 3.27% Acid Yellow 23) 

Re-registration 

Kendra Tyler 

Special Review and Reregistration Division , 

Jim Goodyear, Biologist 

and 

Silvia C. Terrnes, Chemist I --.^I-- -* 

"a-. ----- Ecological Risk Branch 111 I 

Envir~nmental Fate and Effects Divisi 

THRU: Daniel Rieder, Branch Chief 

Ecological Risk Branch I11 I 

Environmental Fate and Effects Division 

Conclusions , 

The Environmental Fate and Effects Division has assessed the potential risk of using Aquashade 

(23.63% Acid Blue 9 and 2.39% Acid Yellow 23) and other similar end-use products containing 

Acid Yellow 23 and Acid Blue 9 dyes to control algae and submerged aquatic plants in 

ornamental ponds, small lakes, fountains and fish pondslhatcheries. ~asdd on available toxicity 

information with a variety of species, these dyes, separately or when cordbined in various enduse products do not cause significant acute or chronic toxic adverse effecks. There is some 

uncertainty in this generalized conclusion given that we only have testind on two of the 

representative end-use products (Aquashade and Admiral WSP). Most ok the data are from acute 

studies where a single high dose is used, and at which no mortality of otder adverse responses 

I 

Text Searchable File
were observed. One mammalian chronic study with the yellow dye also indicated no adverse 

effects from long-term exposure at levels much higher than expected environmental exposure. 

In addition to registrant submitted data, the EFED also reviewed data obtained from the open 

literature. Effects open literature studies were obtained through the ORD Middle Ecological 

Division's ECOTOX literature search and retrieval program managed in Duluth. The data 

obtained from this source supported the contention that the dyes found in Aquashade and other 

end-use products are practically nontoxic to all organisms tested. Open literature fate data were 

obtained through an in-house literature search. 

The products are applied by hand, by either using an eyedropper, a bottle or water soluble 

packets to dispense a prescribed directly into the water to achieve a concentration sufficient to 

block the sunlight energy such that plants can no longer sustain photosynthesis resulting in 

death. The method of application is such that no movement offsite via drift or runoff is 

expkcted. Therefore, the only organisms exposed are the target plants, the fish, invertebrates, 

aquhtic amphibians dwelling in the treated ponds or the birds, mammals, reptiles or terrestrial 

amphibians that drink or move into the treated water. 

Terrestrial plants were assumed not to be exposed under normal use conditions. One uncertainty 

is that none of the labels preclude use of treated water for irrigation. This uncertainty is low, 

beuause it is assumed these treated water bodies would not normally be used for irrigation. 

However, until such language appears on the label, this route of exposure cannot be ruled out. 

No terrestrial plant data were available, however one or more labels for enh-use products 

corhtaining these dyes warns that emerged plants growing in or near treated water that accidently 

come in contact with these end-use products they may "suffer contact burns" as stated on labels 

suCh as the Pond Care Algae Blocker, Aquashade OA, Aquashade, and Admiral. Therefore, 

EqED would assume any inadvertent exposure to terrestrial plants via irrigation or other routes 

would have adverse affects. 

The EFED estimated concentrations in the water based on the application rates specified in the 

labels to attain target concentrations in the water body. These target concentrations were 

compared with fish and invertebrate toxicity values. The target concentrations specified in the 

labels do not approach the concentrations at which no effects or mortality were observed in 

laboratory tests. These dyes are often used in fish bearing waters (ornamental fish ponds or 

hatcheries) and thus it is assumed, and our analysis corroborates, that these dyes do not adversely 

affect fish. By inference, EFED assumes amphibians would not be adversely affected. These 

dyes are highly hydrophilic and not likely to bioaccumulate in fish. 

As would be expected, aquatic plants in the treated water are adversely affected (killed by 

intent), No quantitative assessment was performed for aquatic plants. 

Endangered Species 

The.EFED also assessed the potential for Aquashade and other end-use products containing the 

~cid'yellow 23 and Acid Blue 9 dyes to potentially cause harm to Federally' listed threatened 

and endangered species. 
Terrestrial Animals: Listed endangered or threatened species of birds, mammals and reptiles are 

not expected to be affected directly because exposure does not exceed the endangered species 

LOC. However, terrestrial animals may be affected indirectly if aquatic plants that are necessary 

for their survival are eliminated. The degree to which this might occur depends on the extent the 

treated water bodies are a critical component of their food supply. 

Terrestrial Plants: Terrestrial plants have the potential for exposure if water treated with 

Aquashade or the other dye containing end-use products are used for irrigation. If irrigation is 

precluded fiom use on the labels, EFED concludes there is virtually no potential for exposure to 

terrestrial plants. 

Aquatic Animals: Listed species of fish, invertebrates or amphibians are not expected to be 

affected directly because the exposure does not exceed the endangered species LOC. However, 

aquatic animals dwelling in treated ponds might have the potential for ham if the plants on 

which they depend for food and habitat are eliminated. The likelihood of this occurring depends 

on if any listed aquatic animal species would occur in these highly managed, often manrnande, 

normally relatively small water bodies with usually no outflow, or if any, a very small amount. 

Aquatic Plants: Listed species of aquatic plants which begin their life stages as submerged might 

be harmed if water bodies in which they are growing are treated while they are submerged. 

The EFED has not fully analyzed the potential for endangered species to be harmed through use 

of Aquashade and other end-use products. Such analysis would focus on the overlap of specific 

species with potentially treated water bodies, and in the case of indirect effects, the likelihood 

that the treated water body comprises enough of a component of the li~ted~species food or habitat 

base to actually impact the listed species, if that food or habitat was lost. 

Significant questions raised by the assessment: 

The following issues were identified in the available data for dyes and products 

I A. Purity of the Dyes and Products. 

The experimental characterization of the physical and chemical properties required under 

FIFRA was not apparently done with a 100% pure dye. The Acid ~lue 9 is reported to be 

50% pure (43503401) and Acid Yellow 23 as being 28% pure (431503402). Although the 

presence of chemical impurities affect the physical and chemical properties of a chemical 

substance, the extent by which they affect those of these dyes is ubown. Some of the 

physical and chemical properties reported in these studies are not !consistent with those 

expected for dyes and these differences may be related to the unspecified impurities. For 

example, the reported vapor pressure is too high and likely to be associated with volatile 

impurities. 

2 The Aquashade product that was used in the "basic six" studies was labeled as 

containing 23.63 % Acid Blue 9 and 2.39% Acid Yellow 23. The study authors stated, 
"The test substance characterization provided by the sponsor indicated a purity of 13.9% 

azure blue dye. The use of the term "azure" is unclear, as it could describe a color or a 

series of structurally related "azure dyes". These azure dyes are not structurally related to 

Acid Blue 9 or Acid Yellow 23. Therefore the exact composition and purity of the 

toxicant that was used in the studies is not certain. The registrant must clarify what is 

meant by "azure blue dye". 

3. The above studies were submitted in support of Aquashade (23.6% Acid Blue 9 and 2.39 

Acid Yellow 23 2.39% registration It is unclear if this percent composition takes into 

account the purity of each dye. In addition, it is not known if the impurities in the test 

substances used in the characterization of physical and chemical properties are the same 

as those declared as impurities in the technical andlor end-use product-(~onfidential 

Business Information). It is unclear if the purity of the dyes in otherproducts is the same 

as in Aquashade. 

Label Recommendations 

The EFED has identified confusing language in the current labels and suggests that these label be 

improved for clarity. Adding the following language is recommended. 

I. Water used for irrigation 

It is not clear if treated water can be used for irrigation. If they are used for irrigation, plant 

studies (conducted with a representative end-use product) would be required. It is recommended 

that all of the labels clearly state that "Treated water is not be used for irrigation " 

2. Outflow of treated water 

Include the following statement in all of the labels: "Apply only to bodies of water that is under 

complete control of the user and have no outJlow " 

C. Status of Data Requirements 

Environmental Fate Data Requirements 

The following Subdivision N Data Requirements may be waived for the currently registered uses 

and products: 

1. [Abiotic] Hydrolysis (1 61 -1) 

2. [Direct] Photolysis in Water (1 6 1-2) 

3. Anaerobic Aquatic Metabolism (1 62-3) 

4 Aerobic Aquatic Metabolism (162-4) 

5. Mobility in SoilsISediments (1 63-1) 
6. Bioaccumulation in Fish (165-4) 

Should additional uses are petitioned andlor new products, the EFED will reevaluate the need for 

environmental fate data. 

Ecological Data Requirements 

No additional testing is being required at this time, except that if the labels are not modified to 

preclude irrigation with treated water, 123-1 or 123-2 terrestrial plant testing would be required. 

It is possible that the seedling emergent study requirement might be llfilled with a Tier I study, 

however, it is likely Tier I1 testing would be required for the vegetative vigor data requirement. 

Table 1. Status of ecological effects data adequacy for Aquashade, dyes and other end-use 

products containing Acid Yellow 23 and Acid Blue 9 dyes. 

Guideline Date Requirements Are Data MRID Study 

Adequate for Ecological Classification 

Risk Assessment? 

71-1 Avlan Acute Oral LDso Yes Acceptable 

Bobwhite Quail 

7 1 -2(a) Avian Subacute Dietary Yes Acceptable 

Bobwhite Quad c 

7 1 -2(b) Avian Subacute D~etary 

Mallard Duck 

Avian Reproduction 

Bobwhite Quail and Mallard 

Wannwater Fish Acute Toxicity LC50 

Bluegill sunfish 

Coldwater Fish Acute Toxicity LCso 

Rainbow Trout 

Freshwater Invertebrate Acute Toxicity 

ECjo Water flea 

EstuarineMarine Acute Toxicity with 

Sheepshead Minnow, Oyster, Mysid 

Shrimp 

Freshwater Fish Early Life Stage 

Fathead minnow 

Freshwater Invertebrate Life Cycle 

Water flea 

Tier I1 Terrestrial Plant Seedling 

Emergence (GF 871) 

Tier I1 Terrestrial Plant Vegetative 

Vigor (GF 871) 

Yes Acceptable 

NA 

Yes 

Yes 

Yes 

N A 

N A 

Reserved 

Resewed 

Testing not required 

~u~plemental~ 

Acceptable 

Acceptable 

Exposure to marine habitats not 

expected 

Chronic effects not expected based on 

mode of action and lack of effects in 

acute tests 

Chronic effects not expected based on 

mode of action and lack of effects in 

acute tests 

~eserved depending labeling precluding 

use of treated water for irrigation. If 

water cannot be used for irrigation, 

exposure to terrestr~al plants unlikely 

See above 
Guideline Date Requirements Are Data MRID Study 

Adequate for Ecological Classification 

Risk Assessment? 

Tier I1 Aquatic Plant Studies 

123 -2 NA Aquatic plants are the target, therefore 

testing is not required Release from 

treated pond to other waterbodies 

considered a vely low probability 

Mode of action suggests low likelihood 

141-1 of toxicity, method of application 

minimizes exposure to terrestrial insects Honey Bee Acute Contact Toxicity 
ECOLOGICAL RISK ASSESSMENT 

Re-registration 

"Acid Blue Y"(Eriog1aucine) and "Acid Yellow 23" (Tartrazine ) Dyes Used together in the 

End-use Products Aquashade, Aquashade OA, Admiral Liquid, Admiral WSP and Pond Care 

AlgaBlocker for Control of Algal Growth and Other Undesirable aquatic Plants 

Acid Yellow 23: 

PC Code Number:110302 

CAS Registry No.: 1934-21 -0 

Acid Blue 9 (trisodium salt) 

PC Code Number: 1 10301 

CAS Regishy No.: 

Trisodium salt: 2650-18-2 

Triarnmonium salt: 3844-45-9 

FD&C Blue 1 

In theTriammonium salt. the counter 

cation is ammonium instead of sodium. 

The nature of the counter cation is not relevant 

because it is the anion what is responsible for 

color of the dyes 

Reviewers: 

Ecological Effects: James Goodyear, Biologist 

Environmental Fate: Silvia C. Termes, Chemist 

Secondary Reviewers: 

Mark Corbin 

Stephanie S yslo 

Brian Anderson J 

Branch Chief: 

Daniel Rieder 

Ecological Risk Branch I11 

Environmental Fate and Effects Division 
Table of Contents ..................................................... I. Executive Summary. ..3 

A. Nature of the Chemical Stressor 

B. Potential Risks to Non-target Organisms 

C. Conclusions to the Exposure Characterization 

D. Conclusions to the Effects Characterization 

E. Uncertainties and Data Gaps 

IV. 

Problem Formulation.. ................................................... .8 

A. Stressor Source and Identity 

B. Receptors 

C. Assessment End-points 

D. Conceptual Model 

E. Measures to Evaluate Risk Hypothesis 

F. Measures of Ecosystems and Receptor Characteristics 

G. Analysis Plan 

Analysis.. .................................................................. -19 

A. Use Characterization 

B . Exposure Characterization 

C. Ecological Effects Characterization 

Risk Characterization.. ................................................... .32 

Literature Cited.. .......................................................... .39 

Appendices 

Appendix A 

Appendix B 

AppenQx C 

Appendix D 

Appendix E 

Appendix F 

Appendix G 

Appendix H 

Appendix K 

Data Gaps 

Environmental Fate 

Aquatic Exposure and Modeling 

Ecological Toxicity and Uncertainty in Ecological Toxicity Data 

Status of Data Requirements 

Environmental Fate Bibliography 

Ecological Toxicity Bibliography 

Endangered and Threatened Species 

Risk Quotient Calculation and Interpretation 
I. Executive Summary 

A. Nature of the Chemical Stressor 

The chemical stressors are the dyes Acid Yellow 23 and Acid Blue 9, which are used 

together in several end-use products intended to control algae and other undesirable aquatic 

plants in artificial, confined water bodies (ornamental ponds, golf course lakes, fountains; 

hatcheries; swimming pools). The dyes block sunlight energy necessary for algal growth and, at 

the same time, impart a greenish blue coloration to the water. The two dyes are also regulated 

I under FIFRA as "inerts" used as colorants in some pesticide formulations. In addition, they are 

regulated under the Food, Drug, and Cosmetic Act as colorants for food (food additives), drug 

formulations, and cosmetics1. 

The specific end-use products containing Acid Blue 9 and Acid Yellow 23 are: (a) Pond 

Care Algae Blocker (2.36% Acid Blue 9 and 0.24% Acid Yellow 23; EPA Reg. No. 8709-6); (b) 

Aquashade (23.6% Acid Blue 9 and 2.39% Acid Yellow 23, EPA Reg. NO. 3306-1); (c) 

Aquashade OA (2.36% Acid Blue 9 and 0.24% Acid Yellow 23; EPA Reg. No. 33068-2); (d) 

Admiral Liquid (15.31% Acid Blue 9 and 1.00 % Acid Yellow 23; EPA Reg. No. 67064-2); (e) 

Admiral WSP (49.72% Acid Blue 9 and 3.27% Acid Yellow 23; EPA Reg. No. 67064-1). Except 

for Admiral WSP, all the products are liquid formulations. 

The Agency had assigned the PC 110301 to Acid Blue 9,110302 to Acid Yellow 23, and 

110303 to Aquashade, the mixture of 23.6% Acid Blue 9 and 2.39% Acid Yellow 23 (an end-use 

, product). In this document, the names Acid Blue 9 and Acid Yellow 23 were used when the 

percentage of each dye was not specified or when individual dyes were dehcribed separately. 

"Aquashade" was used when referring to the test substance used in the ecological toxicity 

studies. 

B . Potential Risks to Non-target Organisms 

Based on available toxicity data and the exposures expected when used according to the 

label, the Environmental Fate and Effects Division (EFED) does not believe that "Aquashade" 

and other products containing Acid Blue 9 and Acid Yellow 23 ' 

The specific names used for "Acid Blue 9"under the FDCA arc "FD&C Blue No.1" for the trispdium salt and "D&C" "Blue 

No.4" for the triammonium salt (also known as erioglaucine). Both are salts of the same anion. Acid Yellow 23 is FD&C 

"Yellows", also known as Tartrazine. 

The ecological toxicity studies were conducted with an Aquashade end-use product containing 23.6% Acid Blue 9 and 2.365 

Acid Yellow 23. The PC Number assigned to the product is 110303, which has created some confusion because this product 1s a 

combination of two dyes, each with and assigned PC Number, 
pose a direct acute risk to freshwater fish and invertebrates, birds, amphibians, reptiles or 

mammals, including endangered species. These dyes are not expected to harm terrestrial animals 

that drink treated water. Note that even though we do not have toxicity data on amphibians and 

reptiles, aquatic phase amphibians are represented by fish, and reptiles and terrestrial phase 

amphibians are represented by birds. It is possible that endangered terrestrial animals may be 

affected indirectly by loss of food in water bodies where treatment occurs. The extremely 

limited area in which this may occur, given the label-specified water bodies and the method of 

application (i.e. by hand) may allow a further analysis to reduce this apparent possibility to be 

almost nonexistent. However, this analysis has not been completed. 

Aquashade is an aquatic herbicide and will kill algae and nontarget submerged aquatic 

plants. If any treated habitats contained endangered aquatic plants at the time of application, 

effects are possible. Exposure to emerged plants and terrestrial plants is assumed to be low, 

resulting in little or no risk. 

The Environmental Fate and Effects Division is unable to assess the potential chronic risk 

to animals due to lack of data and therefore cannot conclude that potential chronic risk to these 

taxa (and the taxa for which they are surrogates) does not exist. However, because of its low 

acute toxicity, mode of action which is not toxicological, and because Acid Yellow 23 dye is 

shown not to be chronically toxic to mammals, EFED does not believe that chronic effects are 

I likely and therefore, chronic studies are not needed. 

C. Conclusions- Exposure Characterization 

Status of Data Requirements 

All of the environmental fate data requirements were placed in "Reserved" in 1993, 

dependng on the results of the required ecological toxicity studies. Because the risk assessment 

did not identify risks to fish, aquatic invertebrates, or mammals, the environmental fate studies 

may be "Waived". The environmental fate in this assessment is qualitative, based mostly on data 

from the open literature on structurally related dyes. Although structure-activity relationship 

(SAR) estimates using EPI SU~~~IEPIWIN~ were performed, these programs do not adequately 

estimate physical and chemical properties of salts, particularly those of a large anion such as in 

Acid Blue 9 and Acid Yellow 23. For this reason, the qualitative assessment relies on open 

literature information. 

No environmental fate data from Subdivision N guideline studies are required for the 

present uses. If new uses and/or changes in rate or method of application or products are 

p~oposed, the need for new data will be reevaluated. 

EPI (Estimation Programs Interface) suiteRV' (formerly known as EPIWIN) 
Exposure in the Environment 

Unlike the uses on food, drugs and cosmetics, the dyes are exposed to an open (but 

contained) aquatic environment when used as herbicides. Because the concentrated products are 

added directly to a water body, the dyes (which do not react chemically with each other) 

become diluted in the treated water body. Neither runoff nor spray drift are routes of exposure 

because a specified amount of product is directly applied to the water body to attain 

recommended target concentrations of the product of either "1 ppm or 2 ppm", depending on the 

weed to be controlled. To attain these target concentrations, the labels specify the volume of 

product to be added per volume of water to be treated. These maximum, target concentrations 

were assumed to be maintained after treatment. That is, no degradation was assumed. 

The major route of dissipation of the dyes in an aquatic environment is likely indirect 

photolysis, which depends of on the nature and concentration of natural photosensitizers as well 

as on the geographical location where and season when the products are used. However, 

biotransformation under anaerobic conditions may also contribute to the dissipation of each dye. 

The specific chemical nature of photoproducts and metabolites is not known. 

The dyes are predominantly associated with the water column and have no potential to 

volatilize from water. Although the dyes are not applied to soils, they are dnlikely to volatilize 

from soils. Acid Blue 9 and Acid Yellow 23 do not have the potential to bbaccumulate in dsh. I 

D. Conclusions- Effects Characterization 

Aquashade is a formulation that contains, as its active ingredients, yellow and a blue 

dye. These dyes, Acid Yellow 23 and Acid Blue 9 are also used as food drug and cosmetic 

colorants for human consumption. Their mode of herbicidal action is not ~oxicological, therefore 

little toxicity is expected. he toxicity information available to characteriie the toxicity to 

wildlife includes testing with formulations such as Aquashade and Admiral WSP and also testing 

with the individual dyes. Data used to characterize effects include studied conducted by a 

registrant (testing with Aquashade and Admiral WSP) and studies reported in published 

literature. Published literature were obtained by the Office of Research and DevelopmentIs 

(ORD) Middle Ecological Division in Duluth through their literature searphed conducted as part 

of the ECOTOX program. All testing indicates that these dyes, whether alone, or in formulations 

cause no toxicity to fish, aquatic invertebrates, birds or mammals at doses and concentrations far 

above those expected in the environment. No studies were required or fobnd that tested tde dyes 

or formulations on aquatic or terrestrial plants. In as much as this is a control agent for 

submerged aquatic legetation and algae, it is assumed to have an advers4 effect on aquatic 

plants. The label also warns that if it gets on emergent vegetation, some burning will occur. 

This indicates that these dyes and formulations may be toxic to terrestrial plants. However, the 

method of application would preclude exposure to kestrial plants, so t&city to this group of 

organisms is not considered necessary. If treated waters are used for irrikation, exposure to 

terrestrial plants may be possible and toxicity testing for terrestrial wohld be needed to 

assess risk. 
E. Uncertainties and Data Gaps 

1. Exposure 

Exposure to the dyes were based on the target dilution concentration of a directly applied 

volume of product to a volume of water body to attain the concentrations specified in the labels. 

(1 or 2 ppm; 1 or 2 mg/L). Thus, the exposure concentrations in water are the same as the target I 

concentrations in the labels. It is assumed that the dyes are stable in the water and that the target 

concentration remains constant. That is, routes and rates of dissipation were: not taken into 

account, as data are not available. I 

Indirect photolysis has been identified as the major route of dissipation of the dyes in 

aquatic systems. However, the specific nature of transformation products fdr Acid Blue 9 and 

Acid Yellow 23 is not known. Although no kinetics data are available to asless how fast each 

dye photodegrades, the geographical location, season, and naturelconcentration of natural 

photosensitizers would control the rate of photolysis. Therefore, an uncertainty exists on the 

chemical nature and concentration of photoproducts. In addition, anaerobii biotransformation 

may also be a route of dissipation, but the chemical identity of the metabolites specific to each 

dye is not known. 

Purity of the Dyes 

Three major issues have been identified that are associated with the purity of the dyes 

, and/or test substance used in the ecological toxicity studies. 

a. The experimental characterization of the physical and chemical properties required under 

FIFRA was not apparently done with a 100% pure dye. The Acid Blue 9 is reported to be 50% 

pure (43503401) and Acid Yellow 23 as being 28% pure (43503402). Although the presence of 

chemical impurities affect the physical and chemical properties of a chemical substance, the 

extent by which they affect those of the dyes is unknown. Some of the physical and chemical 

properties reported in these studies are not consistent with those expected for dyes and these 

differences may be related to the unspecified impurities. The high vapor pressure reported in the 

studies may be that of a volatile impurity. I 

b. The above studies were submitted in support of Aquashade (23.6% Acid Blue 9 and 2.39 

Acid Yellow 2.39% registration It is unclear if this percent composition take into account ithe 

purity of each dye. In addition, it is not known if the impurities in the test substances used in the 

characterization of physical and chemical properties are the same as those declared as impurities 

in the technical and/or end-use product (Confidential Business Information). It is unclear if the 

purity of the dyes in other products is the same as in Aquashade. 

c. The Aquashade product that was used in the "basic six" studies was labeled as containing 

23.63 % Acid Blue 9 and 2.39% Acid Yellow 23. The study authors stabed that the test 

substance characterization provided by the sponsor indicated a purity of 13.9% "azure blue dye". 

The use of the term "azure" is unclear, as it could describe a color or a series of structurally 
related "azure dyes". These azure dyes are not sthcturally related to Acid Blue 9 or Acid Yellow 

23. Therefore the exact composition and purity of the toxicant that was used in the studies is not 

certain. The registrant must clarify what is meant by "azure blue dye". 

2. Effects 

EFED has toxicity data for both formulations containing the yellow and blue dye, plus 

testing with the dyes separately. Tests with the Aquashade product containing 23.6% Acid Blue 

9 and 2.36% Acid Yellow 23 as the test substance were conducted on birds, fish and aquatic 

invertebrates. The Health Effects Division (HED) has a rat study conducted with Admiral WSP, 

which showed that the LDS0 was equal or higher than 5,000 mg/kg. There are also mammal 

acute toxicity data with both the blue and yellow dyes also indicating low toxicity. 

No chronic studies using aquashade or other formulations with animals have been 

submitted, so it is not known for certain what would happen over an extended exposure period. 

However, there is mammal chronic testing with the Acid Yellow 23 dye which indicates it does 

not have chronic effects on mammals. Furthermore, these dyes are used as colorants in human 

foods drugs and cosmetics, so long term effects from either the yellow or blue dye to mammals 

are not expected. While directly correlating lack of long-term mammal toxicity to birds is 

uncertain, given the mode of herbicidal action and low level of exposure it (is considered unlikely 

that long-term exposures would have adverse effects on birds. The effects lof chemical 

impurities in thedyes is not known. 

No studies on aquatic plants have been required, because they are the target of 

Aquashade and other end-use products and it is assumed that all submerged aquatic plants in the 

treated pond will be killed. No terrestrial plant studies have been required, because there is no 

expected exposure unless treated water are used for irrigation. 

the 

not 

Because of the mode of action (blocking sunlight energy that enters bodies of water) and 

low acute toxicity of this pesticide, it is believed that additional ecological toxicity studies are 

needed unless new uses, methods of applications or new products are proposed. 
11. Problem Formulation 

A. Stressor Source and Distribution 

1. Source and Identity of the Stressor 

Pond Care Algae Blocker, Aquashade, and Admiral are trade names for products 

containing the two dyes4 Acid Blue 9 and ~cid Yellow 23 which do not react chemically with 

each other. Each product has a different ratio of the dyes (see below), but in all of the product 

formulations the percent of Acid Blue 9 is higher than that of Acid Yellow 23. The use pattern 

of the products are essentially the same in the sense that all of the products are applied directly 

(as a volume of product or number of packets) to artificial (or natural), confined water bodies, 

including swimming pools (provided that chlorine disinfectants are not used). Minimal or no 

outflow are specified in the labels because the mode of action is such that allowing treated water 

to outflow would eliminate the effectiveness of the products. These dyes must remain in the 

water column and block the sunlight energy for a sufficient time to inhibit photosynthesis and 

cause the plants to die. They are not intended for use in non-confined natutal water bodies, 

drinking water sources, or artificial water bodies with outflow. Use of the treated water for 

irrigation is not precluded on the label, and this is an uncertainty because irrigation could result 

in exposure to nontarget terrestrial plants, and there are no tests with terrestrial plants. 

The specific names of the end-use products containing Acid Blue 9 and Acid Yellow 23 

as the active ingredients are AlgaeBlocker and Aquashed OA (both 2.36% Acid Blue 9 and 

0.24% Acid Yellow 23), Aquashade (23.63% Acid Blue 9 and 2.39% Acid Yellow 23), Admiral 

Liquid (15.31% Acid Blue 9 and 1.00% Acid Yellow23), and Admiral WSP (49.72% AcidBlue 

9 and 3.27% Acid Yellow 23). Additional information on these end-use psoducts is presented 

within the "Use Characterization" section. 

The dyes Acid Blue 9 and Acid Yellow 23 are known by several qfferent names, which 

may cause confusion about the chemical identity of the dye. For this reason, each one is also 

identified by the chemical name used by the Office of Pesticide Programs, the Chemical 

Abstracts Registry Number, and their synonyms (Table 1). "Acid Blue 9'; could refer to either 

the trisodium and triammonium salts, each one having a different Che$cal Abstracts Registry 

Number. In most of the product labels, the blue dye is identified as "Acid Blue 9" alone, without 

indicating which salt is used in the formulation. Throughout the documen&, the names "Acid Blue 

9" and "Acid Yellow 23" are used, as they are identified as such in the prbduct labels. In the 

Admiral products, they are identified as "tartrazine" for the yellow dye and as "erioglaucine" for 

the blue dye. The nature of the counter cation for Acid Blue 9 in the products is unclear, 

although the Color Index International identifies it as the triammonium salt. It is likely that the 

blue dye used in the products is the triarnrnonium salt. Further informatibn on the chemical , 

identity of the dyes are included in Table 2. However, because it is the adion of the blue dye 

what exerts herbicidal activity by blocking sunlight energy, the identity df the counter cation is 

not relevant to the exposure and risk assessment when used as herbicidesl. 

A dye is defined as a chemical compound that is capable of imparting color and that is soluble in the vehicle in which it is 

applied. 
In addition to being regulated under the Federal Insecticide, Fungicide, and Rodenticide 

Act (FIFRA), the two dyes are regulated by the Food and Drugs Administration (FDA) under the 

Food, Drugs, and Cosmetics Act (FDCA) as color additives (colorants) in food, drugs and 

cosmetics . Acid Yellow 23 (FD&C Yellow 5) is a food, drug, and cosmetics colorant. The 

trisodium salt of Acid Blue 9 is a FD&C colorant under the name "Blue I", whereas the 

triarnmonium salt (Blue 4; erioglaucine) is a colorant in drugs and cosmetics (D&C). Use on 

foods, drugs, and cosmetics require batch5 certification prior to use. While the nature of the 

counter cation is important for regulation under FDCA, under FIFliA the pesticide active species 

is the anion. It is the anion what gives color to the treated water. Therefore, for the use of these 

blue dyes as an algicide and aquatic herbicide for submerged plants, the nature of the counter 

cation is irrelevant. While the use of the dyes in food, drug formulations and cosmetics require a 

certification of their purity, the purity of the dyes used as an algicidel aquatic herbicide is 

unclear. 

Acid Blue 9 and Acid Yellow 23 are also regulated under FIFRA as "Inerts" used to 

impart color to pesticide formulations. On December 21,2004 the Agency completed the 

"Reassessment of the Exemptions from the Requirement of a Tolerance for the FDA-Certified 

FD&C Additives Blue No. 1 [Acid Blue 91; erioglaucine] and Yellow 5 [Acid Yellow 23; 

tartrazine]" 6. This reassessment was based on structure-activity relationships (EPISuite) and 

does not include a discussion of transformation products.. However, physical and chemical ' 

properties and environmental fate estimates using EPIWIN are uncertain because salts of large 

anions are not adequately handled by EPIWIN. 

The Society of Dyers and Colourists and the American Association of Textile Chemists 

and Colorists have developed a Color Index international7 standard for iddntification of 

pigments and dyes. The Color Index classifies dyes and pigments by theircomposition. 

Individual pigment or dye are identified by a unique Color Index Generic pame (C.I. Name) and 

a Color Index Constitution Number (C.I. Number), although the C.I. Generic Name is most 

commonly used.. I 

Expanded listings of other names used for Acid Yellow 23 and Acid Blue 9 can be found 

from the Unilever Center for Molecular Informatics, Cambridge University, 

"Batch" is defined as "an homogeneous lot of color additive or color additive mixture produced by an identified 

production operation, which is set apart and held as a unit for the purpose of obtaining certification of such 

quantity". The "Batch Number" is the number assigned to a batch of colorant by the person who requests 

certification of the batch. A "Lot Number" is the identifying number or symbol assigned by the FDA to a batch of 

color additive after certification. Note than only the trisodium salt (Blue 1) can be used in food. 

"~eassessmnt of the Exemption from the Requirement of a Tolerance for the R)A-Certified qlor Additives FD&C Blue 

No.1, FD&C Red No. 40, and FD&C Yellow No. 5 (Tartrazine)". U.S. Environmental Protection Agency, Office of Pesticides 

Programs. December 21,2004. 
Acid Blue 9: http://www .dspace.cam.ac.uklhandle/l8 10157 13 

Acid Yellow 23: http://www.dspace.cam.ac.uklhandle/l8 101613 1 

Table 1 Chemical Nomenclature and Structure of the Dyes 

5-hydroxy-1-(p-sulfopheny1)-4-((psulfophenyl) azo) pyrazole-3-carboxylic 

acid trisodium salt 

~olecular Structure 

Acid Blue 9 

Erioglaucine (diammonium salt) 

N-Ethyl-N-(4-[(4- {ethy1[(3- 

sulfophenyl)methyl]arnino)phenyl)(2- 

sulfophenyl)methylene]-2,5- 

cyclohexadien-1-ylidenel-3- 

sulfobenzenemethanaminium 

hydroxide inner salt, trisodium salt 

Common Name as Appearing in the 

labels of the Admiral Products 

Chemical Name(s) 

i-~th~l-~-(4-[(4-{eth~l[(3- 

sulfophenyl)methyl]amino)phenyl)(2- 

sulfophenyl)methylene]-2,scyclohexadien-1-y1idene)-3- 

sulfobenzenemethanaminiurn 

hydroxide inner salt, triammonium 

Acid Yellow 23 

Tartrazine 

4,5-Dihydro-5-oxo-l-(4-sulfophenyl)-4- 

((4-sulfopheny1)azo)-1H-pyrazole-3- 

carboxylic acid trisodium salt 

3~c~~boxy-~-hy~o~y-l-p~sulfophenyl-4- 

p-sulfophenylazopyrazole trisodium salt 

silt 

Only the structure of the disodium 

salt is represented. The diammoniurn 

salt has ammonium instead of sodium 

as the counter cation. It is the organic 

anion what is responsible for the color 

of the dye. 

,sq Na+ 
CI Food Yellow 4 

CI Number 15985 

CAS Registry No. 1934-21-0 

Sodium Salt: 

Brilliant Blue 

CI Food Blue 2 

Erioglaucine trisodium salt 

CI Food Blue 2. 

CI Number 42090 

CAS Registry No. 2650-18-2 

Synonyms 

Triammonium salt 

Erioglaucine diammonium salt 

Alphazurine 

CI Blue 8 

CI 42090 

CAS Reg. NO. 3844-54-9 

FD & C Yellow No. 5: 

21CFR74.705 (Foods) 

21CFR74.1705 (Drugs) 

21CFR74.2705 (Cosmetics) 

These FD&C dyes are of high purity and 

require batch certification analysis when 

used in food, drugs, or cosmetics. 

FD&C Blue 1 

21CFR74.101 (Foods) 

21CFR74.1101 (Drugs) 1 21CPR74.2101 (Cosmetics) I 

Other uses of the dye-s include; 

Acid Yellow 23- Dye for wool and silk; As indicator for chlorine estimations in biochemistry 

Acid Blue 9- Biological stain; Textile dye; Wood stain; Indicator 

2. Physical and Chemical Properties and Environmental Fate of the Stressor 

Physical and Chemical Properties 

D&C Blue 4 

2 lCFR74.1104 (Drugs) 

21CFR.74.2104 (Cosmetics) 

Chemical Family Aminotriphenylmethane dye 

The most distinct characteristic of dyes is that they absorb energy8 strongly within the 

wavelength range of the visible spectrum (360 to 750 nm) and that they are highly soluble in the 

vehicle in which they are used, in this case water. Dyes are highly conjugated systems (i.e., 

multiple double bonds), for which multiple resonance structures can be written. These resonance 

structures cause shifting or appearance of absorption bands into the visibte spectrum of 

electromagnetic radiation. Multiple resonance structures increase the stability of a molecule. 

The color of a dye or other colored materials is determined by the energy (wavelength) of 

maximum absorption (electronic absorption) of incident light by the molecule. The observed 

color is determined by the wavelength region in which the substance doe$ not absorb li ht (i.e., F the reflected color). In an "acid dye", the chromophore is part of a negatike ion (anion) . Therefore, the color is independent of the counter cation. Table 2 summdrizes information on the 

light absorption characteristics of each dye. 

Light is energy. The color observed in dyes (or in gems) is caused by electronic transitions between I energy levels of a 

molecule. The color-giving absorption band is known as a chromophore ("color bearer") 

Azo (monoazo) dye; Contains one azo 

group, - N=N-) 

Although Acid Blue 9 and Acid Yellow 23 belong to different chemical families, both dyes co,ntain sulfonate groups. 
Table 2- Electronic Absorption Spectra of Acid Yellow 23 and Acid Blue 9 in Water 

I Data Source I I 

Table 3- Physical and Chemical Properties of Acid ~eilow 23 and Acid Blue 9 

I Marmion. Handbook of U.S. Colorants- Food, Drugs, and Medical Devices, Third Edition.,: 1991. 1 

Acid Blue 9 

620- 639 range; Amax, 630 i 

Blue 

Electronic Absorption Spectra 

Wavelength of Absorption 

Maximum (Amax, in nm) in 

water 

Reflected Color 

I 

EPI Suite, 2004= Tolerance Reassessment Document 

The physical and chemical properties of Acid Yellow 23 and Acid Blue 9 are summarized in 

Table 3. 

Acid Yellow 23 

420- 430 range, A,, 425 

Yellow 

Acid Blue 9, 

C37H34N2Na2 O9 S3 (disodium salt) 

C~~H~~NZ(NH~>Z 09 S3 

(diammonidm salt) 

I 

792.84 (disodium salt) (Marmion) 

782.96 (diarnmonium salt) 

Reddish-violet powder 

(~armion)l 

2.97 x IO-~~ 

there is some uncertainty in these 

estimated dalues, however they do not 

significantly impact the assessment as 

the vapor pressure are very low 

EPI Suite, 12004 

, 

28.6 (Reported in 43503401) 

1.45 mgm, EPI Suite, 2004 

"~om~let~l~ soluble" (Reported in 

43503401) 

I 

-1.50 ' 

EPI Suite, 2004 

and SRC ~ 

I 

(Estimated); Intrinsic 

Information 

Empirical Formula 

Molecular Weight 

Physical State, as pure chemicals 

Vapor pressure,at 25" C, in mmHg 

Solubility in Water, 25 T in 

mg/L 

Log n-OctanollWater Partition 

Coefficient 

Submitted studies= 40503401 (Acid Blue 9) and 40503402 (Acid Yellow 23); According to the stud~es, the purity of Acid 

Blue 9 was 50% (43503401) and that of Acid Yellow 23 was 28%. Thus, the reported physical chemical properties do not 

reflect those of the pure materials.. The high vapor pressure reported for the two dyes are hkely) to be associated with volatile 

impurities. I 

properties 

Acid Yellow 23 

CI&~N~N~~O~SZ 

534.37 (Marmion) 

Bright orange-yellow powder 

(Marmion) 

7.43 x 10‘" 

there is some uncertainty in these 

estimated values, however they do not 

significantly impact the assessment as 

the vapor pressure are very low 

EPI Suite, 2004 

25.0 (Reported in 43503402) 

1 x lo6; EPI Suite, 2004 

"Completely soluble" (Reported in 

43503402) 

-10.7 

EPI Suite, 2004 

and SRC 

SRC= 2004. Syracuse Research Corporation. Interactive Physical Roperties (PHYSPROP) Database ~(rno. &arch terms: FD&C Blue. 

No. 1, m>&C Red No. 40, FD&C Yellow No. 5. (November 28, 2004); http://www.syrres.comlesclphysdemo.htm 

I 

I 
Dissociation Constant (pKa): Acid dyes are weak acids. The pK, values for Acid Blue 9 and 

Acid Yellow 23 are below 4. Therefore, they are completely dissociated in the environmentally 

significant pH range of 5 to 9. 

Environmental Fate Parameters (Extrinsic Properties) 

Dyes such as Acid Yellow 23 and Acid Blue 9 are not readily biodegradable under 

aerobic conditions nor degrade via abiotic hydrolysis (no hydrolyzable groups)10, Although they 

absorb energy strongly in the visible range of sunlight, they appear to undergo slow direct 

photolysisll Direct photolysis would be only significant in clear, shallow water. But dyes have 

varying susceptibility to fading when exposed to sunlight. Light fastness12 is the degree to which 

a dye resists fading, which varies from dye to dye. Indirect photolysis is likely to be the cause of 

fading in water and one of the major routes of transformation in the environment. Unlike uses in 

food, drugs, and cosmetics, the dyes when used as algicides, are exposed to sunlight in an open 

aquatic environment. Given that sunlight energy varies according to latitude and time of the year, 

their rate of fading are expected to vary depending on geographical location and season. 

Bioitransformation under anaerobic conditions has as also been documented for other structurally 

reldted dyes. Acid Blue 9 and Acid Yellow 23 do not react chemically wid each other. 

Although environmental fate properties for Acid Blue 9 and Acid Yellow 23 were 

estimated with Structure-Activity Relationships using the EPI (Estimation Programs Interface) 

suiteTM (formerly known as EPIwIN)'~ , it was considered that the estimates are not reliable 

because EPIWIN is not suitable for handling salts, particularly of those ~4th large anions. 

Therefore, EPIWIN introduces a high degree of uncertainty for dyes. In addition, EPIWIN does 

not provide any information on the products that may form as the result of photoreactions or 

anaerobic biotransformation. 

The vapor pressure and the Henry's Law Constants of dyes are very low and it is unlikely 

that they volatilize from soil or water. Dyes such as Acid Blue 9 and Acid Yellow 23 are very 

hydrophilic (Log Kow << 1) and, therefore , they are not likely to bioaccumulate in fish. 

''~~nch, D.G. "Estimating the Properties of Synthetic Organic Dyes", in Handbook of Property Estimation Methods for 

Chemicals- Environmental Health Sciences, Edited by Robert S. Boethling and Donald Mackay. Published by Lewis 

Publishers, Boca Raton, FL, 2000; Pages 447- 467. And pertinent references therein. 

11 Even though it has the necessary condition (i.e., absorb energy within the spectrum of sunlight) to undergo direct photolysis 

that process is slow. However, they can degrade via indirect photolysis. For further information on indirect photolysis refer to 

Footnote 17. 

l2 1n artist's color language, a dye or pigment that fades is said to be. "fugitive" and those that dy e are called "permanent 

colors". 

l3 http://www.epa.gov/opptintr/exposure/docs/episuite.htm 
3. Pesticide Type, Class, and Mode of Action 

There are five products that contain both Acid Blue 9 and Acid Yellow 23 in their 

formulation: (1) Care Pond AlgaeBlocker (one label); (2) Aquashade (two labels) and (3) 

I Admiral (two labels). The products are used to control the growth of algae in ornamental ponds, 

recreational man-made (closed-system ponds, such as golf course lakes), rearing lakes for nonedible fish., and fountains containing fish. The dyes do not directly kill the algae, but prevent 

growth by blocking sunlight energy necessary for photosynthesis and survival of the algal and 

other undesirable aquatic plants. Thus, these products serve as algicides or as herbicides for 

undesirable aquatic plants. In addition, the dyes confer a bluish green coloring to the water body. 

4. Overview of Pesticide Usage 

These products are to be applied directly to the water body by adding a recommended 

amount of the product to a specified volume of water to attain 1 or 2 ppm according to the target 

weed. The extent of use, timing of application, and where it is used is not known, but 

geographical location and season will affect the rate of fading and frequency of application! The 

dose to be apply varies with the products and is specified in the labels and takes into account the 

volume and depth of the water to be treated. The specific times of application are not included in 

the labels. However, some of the labels recommend that the product be applied "early in the 

season" (presumably Spring) and also indicate that the product can be added to an ice surfice of 

the water body (i.e., before melting). 

B. Receptors 

1. Aquatic Effects 

For aquatic ecosystems, ecological receptors include all aquatic life (fish, amphibians, 

invertebrates, plants) and those terrestrial animals (e.g., birds and mammals) that consume water 

andlor aquatic organisms. Since these products are directly applied to contained water bodies, 

any effects on aquatic life and terrestrial animals would come from consumption of water or fish 

in rearing lakes or fountains. Immersion in the treated water may also have effects, such as 

irritation or changes in color caused by the dyes. Terrestrial animals may' also be affected; 

indirectly if they depend on the aquatic plants in the treated water bodies for food. 

The labels for these products state that non-target aquatic plants (water lilies, hyaqinths, 

cattails) may suffer contact burns if the chemicals are accidentally poured directly into them and 

that desirable submerged plants may also be affected due to reduced levels of sunlight. 

Aquatic organisms that depend on sunlight for survival may be affected by the reduction 

of sunlight. Examples include non-target plants, animals that consume nbn-t&rget or target plants 

or prey on the animals that consume aquatic plants. 

Risk to aquatic animals is based on registrant submitted acute, diletary laboratory studies 

with aquatic vertebrates (rainbow trout and bluegill sunfish) and invertebrates (water fleas). I 
The taxa evaluated for ecological effects are presented in Table 4. 

Mammals 

Table 4. Taxa evaluated for ecological effects in screening level risk assessments. 

Freshwater fish 

Freshwater invertebrates 

Taxon 

Bobwhite quail (Colinus virginianus) 

Laboratory rat (Rattus nowegicus) 

Surrogate Species Used in Risk Assessment 

Bluegill sunfish (Leopomis macrochirus) 

Rainbow trout (Oncorhynchus mykiss)) 

Water flea (Daphnia magna) 

Birds " Mallard duck (Anus playtrhynchos) 

Estuarinelmarine fish Not Required because estuarine exposure is considered 

unlikely 

Estuarinelmarine invertebrates Not Required because estuarine exposure is considered 

unlikely 

Terrestrial plants Not Required because exposure to terrestrial plants is not 

expected providing treated water is not used for irrigation 

Aquatic plants and algae Not Required because aquatic plants are the target and assumed 

to be controlled by use of aquashade 

" a Birds are used as surrogates for amphibians (terrestrial phase) and reptiles. 

b Freshwater fish may be surrogates for amphibians (aquatic phase). 

Because of the very low exposure potential and mode of action and very restricted use 

pattern, only the avian oral, avian dietary, and acute freshwater studies were required. An acute 

rat study was submitted to OPP's Health Effects Division (HED) and E~D used their review as 

a surrogate for wild mammals. 

2. Terrestrial Effects 

Terrestrial plants are not receptors for the direct application of Aquashade to contained 

water. Terrestrial animals (e.g. birds and mammals) may become receptors if they consume or 

. immerse in the water treated with these dyes. 

Risk to terrestrial animals will be based on registrant submitted acute laboratory tests 

with birds (bobwhite quail and mallard duck) and acute and chronic tests, with mammals to 

represent all terrestrial vertebrates. In addition, effects data from open literature will also'be 

considered. I 

3. Ecosystems at Risk 

All of the products are directly applied to relatively small, ornamental or decorative water 

bodies without outlets and are not to be used in or discharged to streams or rivers. The 

ecosystems at risk are those aquatic habitats in the treated water bodies and the areas occupied by 

terrestrial animals that might consume, or immerse themselves in, the treated water. The labels 

allow treatment of ponds in golf courses, which suggests that "natural looking" ponds may be 

treated. However, it is assumed that these water bodies are managed regularly for esthetic 
reasons (i.e. to control weed population and to add color to the water body). The terminology 

"relatively small" is intended to differentiate the potential use areas from extremely large water 

bodies (e.g. large lakes and rivers). These products are used to curtail photosynthesis in weeds 

and algae by blocking sunlight energy and therefore, use of these products to control aquatic 

plants and algae in extremely large or flowing water bodies is impractical. 

C. Assessment Endpoints 

Ecological EfSects 

Assessment endpoints are the "explicit expressions of the environmental value that is to 

be protected." Defining an assessment endpoint involves two steps: 1) identifying the valued 

attributes of the environment that are considered to be at risk, and 2) operationally defining the 

assessment endpoint in terms of an ecological entity (i.e., a community of fish and aquatic 

invertebrates) and its attributes (i.e., survival and reproduction). Therefore, selection of the 

assessment endpoints is based on valued entities (i.e., ecological receptors), the ecosystems 

potentially at risk, the migration pathways of pesticides, and the routes by which ecological 

receptors are exposed to pesticide-related contamination. The selection of clearly defined 

assessment endpoints is important because they provide drection and boundaries in the risk 

assessment for addressing risk management issues of concern. 

. . 

reproduction) of individuals and populations of 

birds. 

2. Survival of individual mammals and 

reproductive effects. 

3. Survival and reproduction of'freshwater fish 

and invertebrates 

4. Survival and Growth of Aquatic Plants 

lb. Bobwhite quail and mallard duck subacute dietary LC50. 

lc. Avian reproductive NOAEL (in this case, no avian 

chronic data are available, low chronic toxicity is inferred 

based on low chronic toxicity to mammals) 

2a. Laboratory rat acute oral L;D5~ and reproduction test 

NOAEL. 

3a. Rainbow trout and bluegill sunfish acute LCSO. 

3c. Water flea acute LC50. 

3d. Fish and invertebrate chronic toxicity NOAEL. 

4. Algae and vascular plant EC50 and NOAEL 

5. Survival and Growth of Terrestrial Plants 5. Terrestrial monocot and dicot vegetative vigor and 

seedling emergent EC25 and NOAEL 

LD50 = Lethal dose to 50% of the test population. 

LCso (EC50) = Lethal (effective) concentration to 50% of the test population. 

EC2S = Estimated concentration at which 25% response in tested population would be expected 

NOAEL = Highest dose or test level at which no statistically significant effects were observed. 

Conceptual Model 

1. Risk Hypothesis 

Risk hypotheses are specific assumptions about potential adverse effects (i.e., changes in 

assessment endpoints) and may be based on theory and logic, empirical data, mathematical 
models, or probability models (EPA, 1998). For this assessment, the risk is stressor-linked, 

where the stressors are the release of Acid Yellow 23 and Acid Blue 9 by direct application into 

artificial and confined water bodies (i.e. no outlets) and in accordance . The following 

hypotheses are presumed for this screening level assessment when the products are used in 

accordance with instructions and the precautions stated in the labels. 

Aquashade represents a group of end-use products containing the two dyes Acid Blue 9 

and Acid Yellow 23. They are used to control weeds in contained water bodies (no outflow) by 

blocking sunlight to aquatic weeds and thereby inhibiting photosynthesis. They are applied 

directly to these contained water bodies. The risk hypothesis is that when used as directed, 

Aquashade and the other end-use products may represent a risk to aquatic and terrestrial 

organisms." 

2. Conceptual Model 

Water is treated directly either by pouring the product directly, or by dropping packets. I 

I Aquatic plants and animals are exposed directly. 

Terrestrial animals may be exposed by drinking the treated water or immersing in it. 

Terrestrial plants are unlikely to be exposed unless treated water is used for spray 

irrigation. 

E. Analysis Plan 

1. Preliminary Identification of Data Gaps and Methods 

Enpironmental Fate 

As a first step, the current status of FIFRA's Subdivision N Data Requirements was 

identified for Acid Blue 9 and Acid Yellow 23. The 161-1 (Abiotic Hydrolysis), 161-2 (Direct 

Photolysis in Water), 162-3 (Anaerobic Aquatic Metabolism), and 162-4 (Aerobic Aquatic 

Metabolism) guideline studies were "Reserved" based on low-volume of use of these chemicals 

(when use as pesticides)14, but pending the results of the ecological toxicity studies (Guidelines 

74 through 72). Physical and chemical properties were submitted in 1994 for the individual dyes 

(MRID Numbers 43503401 and 43503402), reviewed, and deemed acceptable However, these 

tv\io studies were not conducted with a high purity dye (50% for Acid Blue 9 and 28% for Acid 

~kllow 23)). These two studies were submitted in support of the Aquashade product of 23.6% 

~(cid Blue 9 and 2.39% Acid Yellow 23. 

In December 2004, the Agency completed a tolerance reassessment for these two 

chemicals when used as inert components in pesticide formulations. ~ost of the environ mental 

l4 Memorandum: "Review of Phase 4 List D Package for Aquashade" (EFGWB # 93-01 19,93-0120; Chemical # 110301 and 

110302; Case # 819437 and 819438; DP Barcode Dl84289 and D184278). Dated February 4, 1993. 
fate information in the document come from structure-activity relationships using computational 

tools (EPIWIN). In addition, a "Robust Summary for Acid Yellow 23 (tartrazine)15 was 

submitted to the Agency under the High Production Volume (HPV) Challenge Program", which 

also relies on EPIWIN estimates. As already indicated, EPIWIN is not a suitable estimation 

program for salts, particularly for salts of a large anion. Physical and chemical properties 

estimated by EPI SuitelEPTWIN have been included in Table 3 and come from the 2004 

tolerance reassessment document. 

Information from the open literature was included to present a more comprehensive, but 

qualitative, assessment of the environmental fate of the two dyes beyond structure-activity 

estimates (Refer to the Exposure Characterization- Environmental Fate and Transport section of 

this document). 

Given that the ecological risk assessment concluded that products containing Acid Blue 9 

and Acid Yellow 23 did not pose risk to non-target organisms, no further environmental fate data 

are required unless new uses or products are proposed. 

Ecological toxicity 

Measures of effects are obtained from a suite of registrant-submitted guideline studies 

conducted with a limited number of surrogatd species and from open literature. The test species 

are not necessarily intended to be representative of the most sensitive species but rather were 

selected based on their ability to thrive under laboratory conditions. Acute measures of effect are 

the concentrations that produce 50% mortality or growth reduction in the test organisms (LCSOs 

and ECsos, respectively). Chronic measures are the no observed adverse effect level or NOAEL 

which is the highest test level at which significant effects were not observ'ed. I 

2. Measures to Evaluate Risk Hypotheses and Conceptual Model 

a. Measures of Exposure from Contaminated Water 

For these products, which are directly applied to the water body in a specified volume of 

the liquid product or number of packets, (no spraying), the concentration of each dye depends on 

the amount added and the ratio of each dye in the products. The amount to be added depends on 

where it is used and the size and depth of the water body. Neither runoff por spray drift are 

routes of exposure. Therefore, simulation models (GENEEC; PRZM an! EXAMS) are not 

applicable to estimate environmental exposure concentrations in the treated water. 

To estimate exposure to terrestrial animals, estimates of water consumption were used to 

derive daily doses of the formulation aquashade and compared to acute toxicity endpoints. 

b. Measures of Effects 

"Robust Summaries & Test Plans: C.I. Acid Yellow 23 (FD&C Yellow 5)", Submitted ti the Agency by the International 

Association of Color Manufacturers on March 10,2004. 
Since Aquashade (23.6% Acid Blue 9 and 2.39% Acid Yellow 23) is composed of two 

dyes that are never used individually as algicide/aquatic herbicide, all of the studies submitted 

were conducted with the end-use product (Refer to the Memorandum cited in Footnote 13). 

Studies submitted to fulfill the "basic six" requirements (71-lb avian oral, 71-2b avian dietary, 

72-lb and 72-ld Freshwater fish acute, and 72-2b Aquatic invertebrate acute toxicity). Based on 

low toxicity and no effects observed at the highest test level in the acute toxicity test, the mode 

of action, the low chronic toxicity of the yellow dye demonstrated to mammals in chronic 

tesfing, and the fact that both of the dyes are used as food coloring for human foods and often 

used in other animal foods with no long-term effects detected there is no reason to believe that 

otlier effects (reproductive, estuarine, marine, etc.) are likely. Chronic studies are not required. 

For the present ecological risk assessment, EFED has used the ecological toxicity data 

from the studies conducted with together with an acute laboratory rat toxicity conducted with 

Admiral WSP submitted to HED. 

In addition EFED has made the assumption that the dyes do not renresent chronic risks 

based on a mammalian chronic study with the Acid Yellow dye 23. I 

, 

c. Measures of Ecosystem and Receptor Characteristics 

Aquashade's and the labels of the other end-use products specificalll~ state that treated 

water bodies are not to be drained into streams, rivers or other water systems, it is assumed that 

exposure to these other water bodies will not occur. The aquatic organisms\that occur in the 

treated water bodies are generally assumed to have been placed there by people that manage 

these decorative ponds and containers. The submerged plants in the ponds are assumed to be the 

target organisms, so the risk to them is not assessed. I 

Since it is never applied to terrestrial environments, exposure to te~strial animals 

assessment estimating residues on terrestrial vegetation and invertebrates was inappropriate. 

Instead calculations of the amount in water consumed by birds or mammals pnking from the 

treatecl body of water were done to derive a dose of Aquashade to birds and mammals in three 

size classes. I 

The receptors addressed by the aquatic and terrestrial risk assessments for Aquashade are 

summarized in Table 4. For aquatic assessments, freshwater fish and invertebrates were studied. 

Estuarine/marine animals are not represented, because Aquashade is not applied to these 

environments and is not expected to drain into them. 

111. Analysis 

A. Use Characterization 

The use characterization of the products containing Acid Blue 9 and Acid Yellow 23 is 

based on the current labels for these products. Table 7 identifies these end-use' products with 
their EPA Registration Numbers, and label date. Except for Admiral WSP, all of the products 

are liquid formulations. The products are directly poured or droppen into water in a specified 

volume of the liquid formulation or number of packets. No'spraying is involved which could lead 

to spray drift or inadvertent exposure to terrestrial habitats. 

 able 7 Identification of the End-use Products Containing Acid Blue 9 and Acid Yellow 23 as 

the Active Ingredients 

Product 

Pond Care Algae Blocker 

Aquashade OA 

I I I soluble packages 1 

Aquashade 

Admiral Liquid 

Admiral WSP 

The use sites and timing of application are summarized in Table 8. Note that the uses are 

limited to confined water bodies with minimal or no outflow, but that the Admiral products has a 

wider range of use sites than AlgaeBlocker and Aquashade. Only the Admiral products have uses 

on swimming ponds, but treated swimming ponds must not be treated with a!chlorine 

disinfectant to avoid fading of the dyes. Likewise, no carbon filters should be used to prevent 

removal of the dyes from water. 

EPA Reg. No 

Registrant 

Label Date 

8709-6 

Aquarium 

Pharmaceuticals, Inc. 

February 6,2004 

33068-2 

Aquashade, Inc. 

February 23, 1993 

3306-1 

Aquashade, Inc. 

1981 

67064-2 

Becker Underwood 

March 14,2000 

67064-1 

Becker Underwood 

August 17,2001 

Composition (by 

weight) 

Type of Formulation 

2.36 % Acid Blue 9 

0.24 % Acid Yellow 23 

Liquid formulation 

2.36 % Acid Blue 9 

0.24 % Acid Yellow 23 

Liquid formulation 

Table 8- Use Sites and Timing of Application for the End-use Products Containing Acid Blue 9 

and Acid Yellow 23 as the Active Ingredient I 

Target pests 

, 

ptrol of growth of 

many algae and 

underwater aquatic weeds 

I 

'Control of growth of 

many algae and 

dnderwater aquatic weeds 

23.63% Acid Blue 9 

2.39% Acid Yellow 23 

Liquid formulation 

15.31% Acid Blue 8 

1.00% Acid Yellow 23 

Liquid formulation 

49.72% Acid Blue 9 

3.27% Acid Yellow 23 

Concentrated blend in 

pre-measured water 

Product 

Pond Care Algae Blocker 

I 

vtrol of growth of 

many algae and 

underwater aquatic weeds 

Control algae and aquatic 

vegetation I and color the 

dater 

Cbntro~ algae and aquatic 

vegetation and color the 

wkter 

I 

I 

Use Sites 

Ornamental Ponds and recreational 

man-made, closed system ponds (Golf 

course lakes); 

Rearing lakes for non-edible fish; 

Fountains containing fish. 

Time of Application 

I 

Not specified 1 

"Reapply as nekded" 

I 
Ideal Settings: 

Shopping malls 

Executive office parks 

Botanical gardens 

Recreational and amusement parks 

Aquashade OA 

Natural and manmade contained ponds 

and lakes including ornamental, 

recreational, fish rearing and fish 

f~ming ponds, golf course ponds 

Fountains 

Aquarium 

Ornamental Ponds 

Re-circulated or artificial waterscapes 

Do not apply directly to streams, other 

natural bodies, or any body of water not 

under control of the user. Do not apply 

to water that will be used for human 

consumption. 

Not specified 

Admiral Liquid 

Admiral WSP 

Recommends application before weed 

growing season. 

May be applied while ice is still 

covering the water body 

Natural or manmade ponds, lakes, 

fountains, fish farms, fish hatcheries, 

golf courses and swimming ponds 

Natural or manmade ponds, lakes, 

fountains, fish farms, fish hatcheries, 

golf courses and swimming ponds 

season for optimum results 

May be applied while ice is still 

Prior to or early in the weed growing 

season for optimum results 

The liquid concentrates AlgaeBlocker, Aquashade, Aquashade OA,and Admiral liquid 

are added directly to water in a specified volume of product according to the volume and depth 

of the water body to be treated. to reach a target concentration. Admiral WSP is also directly 

applied to water in a prescribed number of packets for specific volume of thle water bodies. The 

resulting concentration of the products in water is fixed to "1 or 2 ppm", de@ending on the target I weed.. The dosing specified in the labels are summarized in Table 9 . 

Table 9.- Dose Recommended for the End-Use Products Containing Acid Blue 9 and Acid 

Yellow 23 as the Active Ingredients I 1 Product I Dose I 

Pond Care Algae Blocker Ornamental vonds and fountains less than 2 feet deep: 8 mL 

of product per every 100 gallons of pond water (378 L); 

Fish rearing ponds and large ornamental ponds over 2 feet 

deep: 4 mL for every 100 gallons of water 

Maximum application rate, 1 ppm 
Most of the labels contain precautionary language andlor environmental hazard 

statements, but the language is not consistent across the labels. Table 10 summarizes the 

"Precautionary Language", whereas Table 11 summarizes the "Environmental Hazard 

Statements" as they appear in each label. 

Aquashade OA 

Aquashade 

Admiral Liquid 

Admiral WSP 

L 

"Small water bodies" (unspecified) 

One drop for each gallon of water 

'Larger water bodies" 

1 oz per 1,000 gallons of water 

For controlling submerged weed, the depth of water must be 

2 feet 

Maximum application rate, 1 ppm 

One gallon of product (1 ppm) per acre with 4 foot average 

depth when submerged weeds and algae are growing at 

depths > 2 feet. target pests are leafy pondweed,-Chara, 

slender naiad, filamentous green and blue algae, spyrogyraWater milfoil. 

2 ppm to prevent production of Hydrilla's tubers. 

The amount of the product added depends on the volume of 

the water body. Amount to be added (volume) per volume of 

water body is specified in the label. 

Regardless of the amount added and volume of the water 

body, the concentration regime in the water is : 

1 ppm for filamentous blue and blue algae, Chara, Leafy 

Pond weed, spirogyra, slender naiad, and water milfoil 

2 ppm is used to prevent Hydrilla tuber production after a 

herbicide treatment 

Same as Admiral Liquld 

Table 10 Precautionary Language Included in the End-use Products Containing Acid Blue 9 and Acid Yellow 23 as the 

Active Ingredients 

Products 

Pond Care Algae Blocker 

Aquashade 

Precautionary Language Included in the Labels I I 

Non-target plants (water lilies, hyacinths, cattails) may suffer 

contact bums if material is accidenially poured directly into 

them 

Desirable submerged plants may also be affected due to 

Aquashade OA 

reduced levels of sunlight I 

I 

None 

Admiral Liquid Water must be under the complete control of the user and 

have little or no outflow (dye concentration must be 

maintained). 

Do not apply directly to streams, or any body of water that 

I may be used for human consumption 

Admiral WSP Same as for Admiral Liquid I 
Table 11 Environmental Hazard Statements Included in the Labels of the End-use Products 

Containing Acid Blue 9 and Acid Yellow 23 as the Active Ingredients 

Products ( Environmental Hazard Statements Included in the Labels 

Pond Care Algae Blocker Do not contaminate domestic livestock, irrigation water and 

streams with outflow. Use only in impounded water with no 

outlet and under the total control of the applicator. Do not use 

in recreational waters intended for swimming or in waters 

Apuashade OA 

I I I 

B. Exposure Characterization 

intended for edible fish 

Desirable plants such as water lilies may suffer contact bum 

if material is accidentally poured on them directly 

Aquashade 

Admiral Liquid 

Admiral WSP 

1. Environmental Fate and Transport 

No experimental environmental fate, Subdivision N Guideline studies were conducted 

with each individual dye as the test substance or an end-use product. These studles were placed 

in "Reserved" pending the results of the required ecological toxicity studies, as indicated in the 

"Analysis Plan" section. For consistency with the tolerance reassessment for inerts, the 

environmental fate assessment originally based findings on estimates from Structure-Activity 

Relationship (SAR) using EPISUITE (EPIWIN). However, these estimates are considered 

uncertain because EPIWIN is not suitable for salts, particularly those of large anions, such as in 

dyes, A literature search for environmental fate studies conducted with Acid Yellow 23 and Acid 

Blue 9 as the test substances did not produced sufficient experimental data specific to these two 

dyes. Therefore, most of the environmental fate information comes from dyes structurally related 

to Acid Yellow 23 (azo dyes) and Acid Blue 9 (triphenylmethane dyes). Fuqher information was 

obtained from the "Handbook of U.S. Colorants- Food, Drugs, and Medical Devices" (Marmion, 

1991). Thus, most of the present environmental fate assessment uses published information about 

the properties of synthetic dyes and other relevant, open literature sources (Lynch, 2000; Lynch, 

D., OPPTS. private communication, August 2005). The environmental fate assessment is only 

qualitative in nature. 

Acid Blue 9 and Acid Yellow 23 belong to different chemical farnilids but both contain 

sulfonate groups. Because none of these two dyes contain hydrolyzable abiotic 

hydrolyqis is not a degradation pathway for these two dyes. 

Shoreline non-target plants (cattals, water lilies) may suffer 

contact burn if material is accidentally poured on them. 

Do not contaminate water by cleaning of equipment or 

disposal of wastes. 

Shoreline non-target plants (cattails; water lilies) may suffer 

contact bum if the material is accidentally poured on them. 

Apply this product only as specified in the label. Do not make 

applications when weather conditions favor drift from nontarget areas. Do not apply where runoff is likely to occur. 

None specified 
As chemical substances absorbing sunlight energy, photodegradation in water can occur 

via direct or indirect photolysis. The direct photolysis of most dyes appears to be slow and would 

be only significant in clear, shallow water (which could be the case for ornamental uses, such as 

in fountains exposed to sunlight). Because the presence of natural photosensitizers in the 

environment16, indrect photolysis has been identified as a major transformation pathway for 

dyes in environmental water. Besides the concentration of photosensitizers in water, the rate of 

photolysis would also be a function of the solar photon flux, which depends on latitude and 

season. For example, photolysis would be faster in the Summer than in the Spring and faster in 

the Southern than in the Northern parts of the country. Acid Yellow 23 appears to be more 

phbtolytically stable than Acid Blue 9, as the latter fades faster (Marmion, 1991). Photodebradation of dyes can also occur on soil surfaces. 

There are no experimental data that identifies the photoproducts of Acid Blue 9 or Acid 

yellow 23. However, for azo dyes (Acid Yellow 23 is an azo dye), azo bond cleavage and 

ph~toredox reactions occur in most cases. Potential products are aminobenzyl sulphonic acids, 

but their specific chemical identity and toxicity are not known. 

In general, most azo dyes appear to resist biodegradation under aerobic conditions. 

However, microbes living in an anaerobic environment can reduce azo bonds resulting in 

significant loss of color (Brown and Laboureur, 1983; Weber and Adarns, 1995; Jank, et al., 

19R8; Baughman, 1995). Some of the products of the anaerobic transformation of azo dyes are 

arovhatic amines. Therefore, in the environment azo dyes can lose their color via photoredoh 

readtions and by anaerobic biodegradation. Anaerobic biodegradation is most likely to occur in 

sedibnents. 

Fading can also occur by reaction with trace metals, such as zinc, tin, aluminum, iron, 

and bopper, mostly via redox reactions. However, fading is primarily a photochemical process. I 

Dyes such as Acid Blue 9 and Acid Yellow 23 are completely dissociated in the 

environmentally significant pH range of 5 to 9. Because the chromophoric species is the anion, 

the dyes do not adsorb strongly to soils or sediments and are predominantlylassociated with the 

wate'r column. Acid Blue 9 has been long used as a hydrological tracer because it does not adsorb 

strongly on soil particulates (Mon et al., 2005; Tsai, et al., 2004) 

The very low vapor pressure and Henry's Law Constant of the days indicate that 

volatilization would not be a significant process. As highly hydrophilic chemicals with Log 

Kow I<< 1, they are not likely to bioaccumulate in fish. 

2. Measures of Aquatic Exposure 

a. Aquatic Exposure Modeling 

l6 Natural photosensitizers in environmental waters include dissolved organic matter (DOM), singlet oxygen (loz), and hydmxy t 

radicals (.OH). These species are photooxidants. In additions, surfaces of semiconducting metal oxides (such as titanium dioxide 

or zinc oxide) may serve as heterogeneous photooxidants and it is a potential technology to treat wastewater containing . dye 

residues. (Baran, et al., 2003). In addition, photoreactions at the surface of iron oxides/hydroxides may also be involved. 
All of these products are to be applied directly to a confined or mostly confined water 

body. Therefore, the concentration of each dye in water is only the result of direct application to 

water and depends on the relative ratio of the two dyes for each product and the recommended 

dose. Neither spray drift nor runoff are routes of exposure for Acid Yellow 23 and Acid Blue 9 

in, the water. A Drinking Water Assessment was perforrned.and it was concluded that there was 

nq exposure from drinking water. This assessment was based on the label restrictions, which 

indicate that treated water is not to be used as a drinking water source for humans. 

For the aquatic assessment, the concentrations of Acid Yellow 23 and Acid Blue 9 were 

those target concentrations specified in the labels (1 ppm or 2 ppm, depending on the target 

weed). These concentrations are attained by directly adding a specified volume of product per 

specified volume of water to be treated (i.e., by dilution of the product into a larger volume of 

water). It should be noted that the dosing language in some labels (e.g., Aquashade OA ) are 

vague ("one drop") in indicating the volume of product to be added to the water body. It is 

unclear if the specified concentration is in terms of percent of product (i.e., the two dyes and 

inerts) nor the purity of the dyes are taken into account.. 

The concentrations resulting from direct application are summarized in Table 12. 

Because there are no kinetics data for indirect photolysis or anaerobic biodegradation, it is 

assumed that these concentrations remain constant. For Acid Blue 9, the estimated 

concentrations do not take into account the type of counter cation (i.e., no molar fraction 

correction, given the uncertainty in the identity of the salt). Even though the environmental 

concentrations were estimated for each individual dye, there are no ecologkal toxicity for the 

individual dyes because the test substance in all of the ecological toxicity studies was an end-use 

Aquashade product containing 23.6% Acid Blue 9 and 2.39% Acid Yello~ 23 of uncertain 

puiity. Therefore, an assessment based on each individual dye was not possible. The ecological 

toxicity of products with a higher percentage of each individual dye andor hifferent ratio of the 

dyes than Aquashade may potentially be under represented. These products are Admiral Liquid 

and Admiral WSP. 

Table 12 Maximum Concentrations of Acid Blue 9 and Acid Yellow 23 Expected from Direct 

Application of Each Product at the Label at Rates Set in the Labels and assuming +00% purity of each 

dye (Source: Product labels) , 

Product 

Aquashade OA 

Aquashade 

Pond Care AlgaeBlocker 

Admiral Liquid 

Application Rate of Product 

as target concentration, ppm; 

1 

2 

1 

2 

1 

1 

2 

Concentration of Acid Blue 

9, in mg1L 

0.024 

0.048 

0.24 

0.48 

0.024 

0.15 

0.3 

- Concentration of Acid 

Yellow 23, in mg/L 

I 

0.0024 

0.0048 

0.024 

0.048 

0.0023 

0.01 

0.02 I 
Admiral WSP 1 0.497 (0.5) 0.033 

I b. Aquatic Exposure Monitoring and Field Data 

The use of monitoring data and/or field data is not applicable to these dyes when used to 

control algal growth or other undesirable aquatic plants in confined, with no or minimal outlet , water bodies. However, these dyes also have industrial applications and have been detected in 

waste water, sewage systems, and water bodies17. Ecological risk from those sources of potential 

exposure are not addressed in this document. 

c. Measures of Terrestrial Exposure 

3. Terrestrial Exposure Modeling 

Routinely, exposure to birds and mammals feeding on a treated field is estimated by i 

modeling residues on terrestrial vegetation and invertebrates using the Terrestrial Residue 

Exposure (TREX) simulation model. Water treated with Aquashade or any of the other products 

is not expected to reach terrestrial vegetation or invertebrates. Because exposure to the dyes from 

residues on terrestrial food items is not expected, the use of terrestrial exposure models is not 

applicable. An exception could be if treated water was used for irrigation. If this happened, it is 

possible the dyes in these formulations would get on terrestrial food items. However, given the 

low toxicity, it is unlikely to result in direct acute risk to terrestrial animals. 

a. Terrestrial Exposure via Consumption of Contaminated Water Modeling 

To determine the exposure of mammals and birds to "Aquashade" via consumption of 

contaminated water, a single daily dose of Aquashade was estimated using the calculated volume 

of water that birds and mammals are expected to consume per day and the concentration of 

Aquashade in water as follows: 

Daily Exposure (mg) = Daily Water Consumption (L) x Water Concentration (mg/L). 

To estimate the volume of water that mammals and birds are expected to consume per 

day, allometric equations from the EPA Wildlife Exposure Factors Handbook (US EPA 1993) 

I were used. For birds, the daily water consumption (L) was calculated using the equation (US 

EPA 1993, Equation 3-15, p. 3-8, for all birds): 

L = a (body weight in kg) B, where a = 0.059 and P = 0.67 

l7 See, for example, "Background Document for Identification and Listing of Deferred Dye and Pigment 

Environmental Protection Agency, Office of Solid Waste, Washington, D.C., June 1999. 

26 
For mammals, the daily water consumption (L) was calculated as using the equation (US 

EPA 1993, Equation 3-7, p. 3-10, for all mammals): 

L = a (body weight in kg) P, where a = 0.099 and f3 = 0.9 

The maximum concentration (weight to volume) of Aquashade in water after application 

at the labeled rate is 2 mg/L. As summarized in Table 13, a single daily exposure via drinking 

water containing 2 mg/L Aquashade was calculated for three weight classes for birds (20, 100, 

and 1000g) and mammals (15,35, and 1000g). 

1,000 0.059 0.119 

Mammals 15 0.0023 0.004 

35 0.0048 0.01 

1,000 0.099 0.1 

a Estimated Daily Exposure (mg) = Daily water consumption (L) x Water concentration 2 ma). 

b. Residue Studies 

There is no terrestrial exposure from this use pattern. Therefore, residue data are not 

necessary. 

C. Ecological Effects Characterization. 

I This document is a screening level evaluation of the potential ecological risk from the 

end-use products that contain Acid Yellow 23 and Acid Blue 9 dyes, and are represented by, a 

formulation referred to as Aquashade. All of the products are composed of two dyes that are 

never used individually as an algicidelaquatic herbicide. These products are applied drectly; to 

confined water bodies and never to terrestrial or aquatic ecosystems. 

Available data are from tests conducted with either a formulation containing Acid Yellow 

23 and Acid Blue 9, or with the dyes separately. 

All of the toxicity studies on birds, fish and aquatic invertebrates submitted by the 

registrant were conducted with the end-use product Aquashade (23.6% Acid Blue 9 and 2.39% 

Acid Yellow 23) and not with the individual dyes. The studies were submitted to support the 

registration of this "Aquashade" end-use product (EPA Reg. No. 33068-1). Through searches 

conducted by the ORD Middle Ecological Division at Duluth under their ECOTOX literature 

27 
search program, ecological toxicity studies conducted with the individual dyes were located. 

The results of these published literature data supported the results of, and did not indicate greater 

toxicity than, or effects other than the registrant submitted tests, therefore, they were not used to 

estimate risk in this assessment. A laboratory rat study was conducted with the Admiral WSP 

product as the test substance (49.62% Acid Blue 9 and 3.05% Acid Yellow 23). 

There is an uncertainty in the purity of the test substance used in the ecological toxicity 

studies. However, considering the low exposure level relative to even the lowest assumed purity, - 

this uncertainty does not cause doubt in the conclusions. 

The studies submitted to hlfill the "basic six" requirements (an avian oral, two avian 

dietary, two fish acute, and one aquatic invertebrate acute toxicity) found very low toxicity 

(practically nontoxic and slightly toxic). A study on the laboratory rat found an LD50 equal to or 

higher than 5,000 ppm. Table 14 summarizes the acute data for the tested species used to assess 

risk. 
Aquashade 

Bobwhite quail Acute Avian Oral 433367-01 LD50 & acute NOAEC 2 2250 

mgm 

Mallard duck Acute Avian Oral 433367-02 LD50 & acute NOAEC 2 2250 

mgm 

Bobwhite quail Avian Dietary 435034-03 LC50 & acute NOAEC 25,620ppm 

Mallard duck Avian Dietary 435034-04 LC50 & acute NOAEC 2 5,620ppm 

Bluegill sunfish Fish Toxicity Bluegill 432975-02 LC50 & acute NOAEC 2 96 mgL 

Rainbow trout Fish Toxicity Rainbow Trout 432975-01 LC50 & acute NOAEC 2 96 mg/L 

Daphnia magna Invertebrate Toxicity 432975-03 LC50 & acute NOAEC 2 97 mg/L 

Test Material 

Admiral WSP 

Laboratory rat Wild Mammal Acute 45281 1-01 LD50 ci & 9 r 5,000 mglkg 

All were perfo~~ned with the Typical End-use Product, Aquashade or Admiral WSP 

* The results refer to the concentration of the end-use product. 

Birds and mammals are expected to drink from ponds that have been treated with the 

end-use products. EEED calculated an estimated dose that they would receive from that water I (see Table 10). 

1. Terrestrial Effects 

Avian effects 

The avian oral acute toxicity studies for both the bobwhite quail and' the mallard duck 

found LD50's and NOAECs equal to or greater than 2,250 mg/kg. The avian dietary toxicity 

studies found LCsos and NOAELs equal to or greater than 5,620 ppm. Aquashade is considered 

to be practically nontoxic to birds. Higher tiered studies are not required. 

Mammalian effects 

An acute toxicity study that used Admiral WSP with the laboratory rAt WAS reviewed by 

OPP's Health Effects Division found an LDso equal to or greater than 5,000 hg/kg. EFED 
considers this to be practically nontoxic. The Health Effects Division wrote that, ". . . this 

formulation contained 49.62 % Acid Blue dye and 3.05% Acid Yellow dye (presumably Acid 

Blue 9 [Erioglaucine], and Acid Yellow 23 [Tartrazine], respectively). A chronic mammal 

toxicity test with Acid Yellow 23 indicated low likelihood of adverse chronic effects at 

environmentally relevant exposure levels. 

Calculating Daily Dose 

Terrestrial animals drink water from ponds and other bodies and may be affected by the 

Aquashade or any of the other end-use products in the water. Exposure estimates for terrestrial 

animals via drinking contaminated water were expressed in terms of a single daily dose (mg) for 

three different body weight classes. All of the exposures were less than 0.2 mg a.i./day. 

Birds 

The weight classes for birds were 20, 100, and 1,000 g. To obtain the toxicity value for a daily 

dose (mg of Aquashade) for each weight class for birds termed the "derived median dose," the 

acute oral LDso (22,250 mgikg-bw) was multiplied by body weight (kg). The calculated daily 

doses for birds for each weight class are summarized in Table 13. 

Mammals 

Exposure estimates for terrestrial animals via drinking of contaminated water were 

expressed in terms of a single daily dose (mg) for three different body weight classes (15,25, 

and 1,000 g). Therefore, to obtain the lethal dose (mg of Aquashade) for each weight class for 

mammals, the acute rat oral LD50 value of 25,000 mglkg-bw (MRID 45281 1-01) was multiplied 

by body weight (kg). The calculated daily doses for mammals for each weight class are 

summarized in Table 15. 

Table 15. Acute toxicity values for birds and mammals drinking from contaminated 

water, expressed in terms of a single Aquashade dose (mg). 

Animals Body Weight (kg) LDSO Derived Median 

(mglkg-bw) Lethal Dose (mg) a 

Birds 

1.000 12,250 22,250 

0.015 25,000 275 

Mammals 

0.035 25,000 2 175 

1.000 25,000 25,000 

a Derived Median Lethal Dose (mg) = body weight (kg) x LD~o (mglkg body weight). 
Terrestrial Plant efSects 

The method of application, i.e. pouring and dropping specific volumes or packets into the 

target water body, is not expected to result in drift or runoff resulting in exposure to terrestrial 

plants. Since it will not be applied to terrestrial plants no plant studies were required. One 

uncertainty exists, however, in that not all the labels preclude use of treated water for irrigation. 

I If it was used for irrigation, the potential routes of exposure would be drift and/or runoff. To 

assess risk to terrestrial plants, tier 1 or tier 2 terrestrial plant testing would be used. If the labels 

I stated that treated water must not be used for irrigation, such testing would be unnecessary. 

Some of the labeling indicates that emergent plants may experience burning if exposed to the dye 

foimulations. Based on this, it is assumed that any inadvertent exposure to terrestrial plants 

might cause adverse effects. 

2. Aquatic Effects 

The registrant submitted studies that determined the toxicity of Aquashade to aquatic 

animals. The bluegill sunfish and rainbow trout studies found LCSOs and NOAECs equal to or 

greater than 96 mg/L. This level is considered by EFED to be slightly toxic. The level for being 

considered practically nontoxic is 100 mg/L for aquatic animals. 

The aquatic invertebrate (Daphnia rnagna) study found that the LCso and the NOAEC 

were equal to or greater than 97 mg/L. This level is considered to be slightly toxic. No further 

studies are required. 

Aquashade, Admiral, and AlgaBlocker are algicides or herbicides for undesirable aquatic 

plants. They are not applied to terrestrial environments nor if treated water used for irrigation. 

Because submerged aquatic plants are the target species and it is assumed that all submerged 

plants will be killed, no aquatic plant studies were required. 

3. ECOTOX and other Published information 

The Agency also obtained summary toxicity information from published literature found 

as part of the ECOTOX program maintained by the EPA Office of Research and Development 

Middle Ecological Division (MED) at Duluth. This program regularly searches open literature 

for toxicity information. OPP requested of MED Duluth to search its holdings for toxicity 

information on aquashade and the dye components of the formulations being assessed for 

reregistration. Toxicity information was located on mammals and aquatic animals from tests with 

either of the dyes. In all cases, the test results located through ECOTOX corroborated the 

findings of the registrant submitted data, and did not indicate effects unforeseen from the 

registrant studies. Neither did these published literature studies indicate adderse/effects to 

organisms other than those tested by the registrant. None of the data located thrbugh ECOTOX 

were used in the assessment. See Appendix F for a summary table of data locat{d from 

ECOTOX. 
IV. Risk Characterization 

, Acid Yellow 23 and Acid Blue 9 are applied together via the end-use products Aquashade 

Admiral Liquid. Admiral WSP, and AlgaBlocker. These products are applied to confined water 

bodies or water bodies with minimal outflow. None of the products have terrestrial uses and 

would not directly contact terrestrial food items for birds and mammals. Therefore, the RQs based 

on dietary exposure from Aquashade residues on food items were not calculated. However, it 

could affect them via drinking water. 

Thus, all of the potential risks of Acid Yellow 23 and Acid Blue 9 are associated with 

direct exposure of terrestrial animals through drinking water or immersion in the treated water. In 

addition, aquatic organisms dwelling in treated water are also exposed to the dyes (refer to 

"Conceptual Model") Risks were estimated based on "environmental" concentrations in a water 

body resulting from direct application of the product(s) that is, a dilution concentrati.on based on 

the application rates indicated in the labels (see Table 10). It must be kept in mind that the 

characterization of effects was based in terms of the combined two dyes for the Aquashade enduse product (23.6% Acid Blue 9 and 2.36% Acid Yellow 23) used in the ecological toxicity 

studies and not for the individual dyes. 

A. Risk Estimation - Integration of Exposure and Effects Data 

1. Birds and Mammals 

Exposure to birds and mammals was expected primarily through consumption of drinking water 

treated with the dyes. Birds are surrogates for reptiles and terrestrial phase amphibians because it 

is assumed for screening level assessment purposes that reptiles and terrestrial phase amphibians 

are not more sensitive to oral toxicity than birds. 

Acute Risk Quotients (RQs) via Consumption of Contaminated Water 

Acute RQs for birds and mammals exposed to Aquashade via consumption of 

contaminated water were calculated for each of three body weight classes using the daily 

exposure value expressed as milligrams of Aquashade (Table 13) and the toxicity value expressed 

in terms of milligrams of Aquashade (Table 14). The RQs are summarized in Table 16. 
1,000 0.12 2 2,250 < 0.01 

Mammals 15 < 0.01 2 75 < 0.01 

" Estimated Environmental Exposure (EEC) = Daily water consumption (L) x Water concentration (mgL). I 

Rounded to two decimal places. 

Derived Median Lethal Dose (mg) =body weight (kg) x LDso (mg/kg body weight). 

Acute RQ = Estimated Environmental Exposure 1 Concentration Derived Median Lethal Dose 

RQs are below the Levels of Concern (LOCs) for acute risk (LOC 0.5), acute restricted use (LOC 

0.2), and acute endangered species (LOC 0.1). 

Acute RQs are below the Levels of Concern (LOCs) for endangered species ( 0.1). 

Use of Treated Water for Irrigation 

I 

While it is considered highly unlikely that treated water would be used for irrigation, 

given the kinds of water bodies treated, if this happened, exposure to birds and mammals might 

occur through ingesting terrestrial food items that had been exposed to the water. Given the low 

toxicity of the dyes, and the relative low concentration in the treated water (no more than 2 ppm), 

it is unlikely that this route of exposure would cause &rect toxicity to birds or mammals. If 2 

inches of irrigation water containing 2 ppm were sprayed onto terrestrial vegetation, the resulting 

"application rate" of the dyes would be approximately 1 Ib ailacre. 

Applying 1 lb ailacre to short grass would result in residues no greaFer than 240 ppm. For 

a small (15g) mammal that eats close to its body weight this would still resblt in a dose per animal 

much less than the lowest toxicity value for mammals. See appendix E forlcalculations. 

2. Non-target Aquatic Animals and Plants 

Aquashade and any of the other end-use product containing Acid Blbe 9 and Acid Yellow 

23 are only applied directly to ponds and other aquatic systems without an outlet. It is not 

expected to come in contact with non-target aquatic animals outside of those systems. 

This is a screening level assessment of the acute risk of using ~~uashade, therefore, the 

highest EECs (2 mgL; for Admiral Liquid and Admiral WSP) and the lowe'?t toxicity values were 

used. The concentration in the treated systems will be higher than any body of water that 

I 

33 I 

I 
accidentally receives (by flooding, etc.) a dose of Aquashade. A summary of aquatic acute RQs 

is presented in Table 17. 

ble 17. Estimated acute risk quotients (RQ) for aquatic animals exposed to Aquashade. 

this screening analysis only the highest Estimated Environmental Concentration (EEC 

I (mgJL) 

Bluegill sunfish 2 96 mg/L <0.01 none 

, Rainbow trout 2 96 mg/L KO.01 none 

Daphnia magna 2 97 mg/L 10.01 none 

Fish are used as surrogated for aquatic phase amphibians, so lack of risk to fish results in a 

conclusion of low risk to amphibians. 1 

Aquatic Plants: This is a screening level assessment based on an Aquashade product. 

Aquashade, AlgaeBlocker, and Admiral products are aquatic algicides or herbicides for 

undesirable aquatic plants and are expected to kill all submerged aquatic plants. However, since 

it is only applied to ponds with little or no outlet, it is not expected to come into contact with 

nontarget aquatic out side of the target pond. All of the submerged plants in the pond are 

considered to be targets. Therefore, the RQs were not calculated for aquatic plants. If the water 

treated by these products were used for irrigation, it is unlikely the concentration of dyes in the 

runoff from such irrigation would be sufficient to cause effects to aquatic plants in receiving 

water bodies. 

3. Non-target Terrestrial Plants in Dry-land and Semi-aquatic Environments 

Terrestrial plants growing in dry-land and semi-aquatic environments are not expected to 

be exposed to the end-use products, since they products are poured, or applied with an eyedropper 

into confined water bodies or water bodies with minimal outflow. Because the labels do not allow 

application to terrestrial ecosystems, a terrestrial plant risk characterization was not performed. 

An uncertainty exists in that the labels do not preclude use of treated water for irrigation. If this 

happened, there is a potential for adverse effects to terrestrial plants because some labels indicate 

that if emerged plants are exposed to the formulations, burning would be expected. 

B. Risk Description - Interpretation of Direct Effects 

Aquashade does not exceed the level of concern for animals with which it is expected to 

come in contact. Because of the method of application, no drift or runoff onto terrestrial habitats 

is expected precluding exposure to terrestrial plants, and to food items consumed by terrestrial 

animals. Thus, the risk hypothesis is proven wrong for these organisms. 

The mode of herbicidal action and intended use will result in adverse effects to target 

aquatic plants, therefore the risk hypothesis that these formulations adversely affkct aquatic plants 

is correct. However, in all cases, the treated water body is treated for the express, reason of 
controlling (eliminating) submerged aquatic plants therefore, this effect is not interpreted as an 

adverse ecological effects unless, as discussed below, there are endangered aquatic plants in these 

treated water bodies. 

C. Endangered and Threatened Species 

The use of these dyes may affect endangered aquatic plants possibly occurring in ponds 

where this might be used. It may also have the potential to affect endangered animal species 

which depend on aquatic plants in these managed water bodies for food and/or habitat. The 

potential for this direct effects to listed aquatic plants, and indirect effects to listed animals is very 

limited because of the method of application (i.e. by hand), and the kinds of water bodies treated 

(golf course ponds and other managed ponds) where keeping the water free of all vegetation is 

aesthetically desirable. These water bodies do not typically have outflow to streams or rivers, 

which would preclude endangered fish and invertebrates from entering. However, endangered 

amphibians may enter such water bodies, and the loss of plants that serve as food and shelter for 

thbse amphibians have the potential indirectly affect these organisms. 

1. Action Area 

For listed species assessment purposes, the "action area" is considered to be the area 

affected directly or indirectly by the Federal action and not merely the immediate area involved in 

the action. At the initial Level I screening assessment, broadly described taxa are considered and 

thus the screening conservatively assumes that listed species within those broad groups are colocated with the pesticide treatment area. This means that terrestrial plants and wildlife are 

assumed to be located on or adjacent to the treated site, and aquatic organisms are assumed to be 

located in a surface water body adjacent to the treated site. The assessment also assumes that the 

listed species are located within an assumed area that has the relatively highest potential exposure 

to the pesticide, and that exposures are likely to decrease with distance from the treatment area. 

Aquashade, Admiral, and AlgaBlocker are used in ornamental ponds, recreational manmade ponds, closed system ponds, golf course lakes, rearing lakes for fish, fountains containing 

fish, and recirculated or artificial waterscapes. Therefore, these products are only applied to 

confined water bodies. 

2. Taxa Potentially at Risk 

In this assessment, it is concluded that the only species potentially at risk are aquatic 

plants from direct effects if they occur in treated ponds and lakes, and aquatic or terrestrial 

animals that depend on the vegetation in the treated water bodies. 

3. Discussion of Risk Quotients 

No direct effects on listed animal species are predicted. No exposure to terrestrial plants 

was expected, so no terrestrial plant RQs were calculated. Even though no RQs were calculated 
for aquatic plants, it is assumed they will be affected, since they are the target organisms to be 

controlled. 

4. Probit Dose Response Relationship 

Since no mortality was observed in the standard studies, no probit analysis was performed. 

5. Data Related to Under-represented Taxa 

The screening level assessment relies on RQ calculations that use toxicity endpoints 

selected from the most sensitive species tested within broad taxonomic groups. There may be 

I situations in which additional effects data from one or more sources may suggest that a given 

suite of listed taxa may be more or less sensitive than suggested by the effects data used for RQ 

I calculations. No such relationships were found in this analysis. 

\ 

6. Implications of Sublethal Effects 

The Level I screening assessment normally relies on the acute mortality endpoint as well 

as a suite of sublethal responses to the chemical stressor. EFED decided that, based on its mode 

of action, the low chronic toxicity of at least one of the dyes to mammals, low potential for 

exposure based on use pattern, and its history as a human food dye, Aquashade did not need 

chronic toxicity studies. Sublethal effects were not analyzed. 

7. Indirect Effects Analysis 

This assessment indicates that Aquashade may impact listed invertebrates, fish and 

amphibians that depend upon submerged aquatic plants and algae. There is also a potential for 

indrect effects to listed terrestrial birds and mammals that occur near treated water bodies, and 

that depend on submerged aquatic vegetation or algae for food or other survival or reproductive 

processes. The likelihood of potential indirect effects has not been quantified or refined. Such 

refinement could incorporate information on specific use locations relative to specific locations of 

listed species. Subsequent refinement may also include additional analyses on potential indirect 

effects to listed species. 

8. Critical Habitat 

Aquashade may impact invertebrates and fish that depend upon the target species 

(submerged aquatic plants and algae). The likelihood of potential indirect effects has not been 

quantified. Subsequent refinement may include additional analyses on potential indirect effects to 

listed species. 

9. Co-occurrence Analysis 

The goal of the analysis for co-location is to determine whether sites of pesticide use are 

geographically associated with known locations of listed species. Listed species potentially at 

risk include aquatic plants, and animals that depend on aquatic effects from indirect effects. 

Aquashade's use pattern makes it (potentially) possible that it will be used in every county in the 
United States: Therefore, EFED's database to find which counties harbor which listed species 

("LOCATES") was not used. However, there are possible effects to 

D. Description of Assumptions, Limitations, Uncertainties, Strength, and Data Gaps 

1. Environmental fate 

Data Source 

The environmental fate assessment is only at the qualitative level. Structure-activity 

edtimations (EPIWIN) of physical and chemical properties/characteristics are uncertain. EPIWIN 

is not suitable for estimating properties of salts of large anions, such as in acid dyes. Therefore, 

1 thts qualitative environmental fate assessment was primarily based on the general, known 

behavior of dyes in the environment and not on the specific behavior of Acid Blue 9 and Acid 

Yellow 23. 

Reaction kinetics and transformation products 

Although indirect photolysis Was identified as a major route of dissipation of the dyes in aquatic 

media, the identity of the photoproducts (and hence, their toxicity) is not known. The rate of 

phototransformation is expected to be a function of geographical location and season. 

Biotransformation under anaerobic conditions may also contribute to the dissipation of the dyes 

and can occur predominately in sediment, but the chemical identity of the metabolites is not 

known. 

Impurities 

The characterization of the physical and chemical properties required under FIFRA were 

not' done with a 100% pure dye. The Acid Blue 9 is reported to be 50% pure and Acid Yellow 23 

as being 28%. Although the presence of chemical impurities affect the physical and chemical 

properties of a chemical substance, the extent by which they affect those of the dyes is not known. 

T~c physical and chemical properties were submitted in support of Aquashade (23.6% Acid Blue 

9 ahd 2.39 Acid Yellow 2.39% registration In is unclear if these percentages take into account the 

pu4ty of each dye. Furthermore, the percent purity of each dye in the other products is unknown. 

Given the low exposure of these dyes in the environment, when used as pesticides, no 

Subdivision N guideline studies are needed for the intended uses. However, if new uses are 

petitioned, the EFED will reevaluate the status of data requirements. 

2 Ecological toxicity 

The assessment assumes that Aquashade and related products will be used strictly in 

accordance with label instructions. It assumes that there will be no large spills of the product 

I 

The ecological toxicity studies were not conducted with the individual dyes, but with the 

mixture of the two dyes as the test substance (23.6% Acid Blue 9 and 2.36 % Acid Yellow 23, An 
Aquashade end-use product). There are two other products that contain a higher percent of the 

dyes than the tested substance, namely Admiral Liquid (1 5.3 1 % Acid Blue 9 and 1.00% Acid 

Yellow 23) and Admiral WSP (49. % Acid Blue 9 and % Acid Yellow 23). Therefore, the 

ecological toxicity of these products is not known. However, the maximum estimated 

environmental concentration resulting from direct application to a water body is below the Level 

of Concern. 

The chronic toxicity RQs cannot be calculated, because there are no studies on chronic 

risk with the pesticide. The low acute toxicity values, along with testing with the Acid Yellow 

dye 23 with mammals, and the mode of action of Aquashade and other products, suggest low 

chronic toxicity and EFED has not required any chronic studies. 

The Aquashade product that was used in the "basic six" studies was labeled as containing 

231.63 % Acid Blue 9 and 2.39% Acid Yellow 23. The study authors stated, "The test substance 

characterization provided by the sponsor indicated a purity of 13.9% azure blue dye." Therefore 

the exact composition of the toxicant that was used in the studies is not certain. The use of the 

wdrd "azure" could describe color, but also the word "azure" could be associated with a series of 

structurally related dyes that are totally different from Acid Blue 9 or Acid Yellow 23. 
V. Literature Cited 

E. Open Literature 

Books 

Lynch, D.G. "Estimating the Properties of Synthetic Organic Dyes", in Handbook of Property 

I Estimation Methods for Chemicals- Environmental Health Sciences, Edited by Robert S. 

Boethling and Donald Mackay. Published by Lewis Publishers, Boca Raton, FL, 2000; Pages 

I 447- 467. And pertinent references therein. 

Marmion, D,M. Handbook of U.S. Colorants- Food, Drugs, and Medical Devices, Third 

Edition., 1991. Published by John Wiley and Sons,'New York 

Helz, G.R., Zepp, R.G., and Crosby, D.G, Editors. 1994. Aquatic and surface photochemistry. 

Lewis Publishers, Boca Raton, Florida. 

Journal Articles 

Jank. M., Kijser, H., Liicking, F., Martienssen, M., and Wittchen, S. 1998: "Decolorization and 

Degradation of Erioglaucine (Acid Blue 9) Dye in Wastewater", Environmental Technology, 

~.19(7), pp.741-747. 

Weber, E.J. and Adarns, R.L. 1995. "Chemical and Sediment-Mediated Reduction of the Azo Dye 

Disperse Blue 79", Environ. Sci. Technol. v.29, pp. 1163-1 170. 

Baughman, G.L. 1995. "Fate of azo dyes in aquatic systems. Part 3: The role of suspended 

sediments in adsorption and reaction of acid and direct dyes", Dyes and Pigments, v.27, pp. 197- 

210. 

Brown, D. and Laboureur, P. 1983, "The Degradation of DyestufSs: Part I- Primary 

biodegradation under anaerobic conditions ", Chemosphere, v. 122, pp. 397-404. 

Baran,W., Makowski, A., Wardas, W. 2003. "The influence of FeC13 on the photocatalytic 

degradation of dissolved azo dyes in aqueous Ti02 suspensions", Chemosphere, v.53, pp 82-95. 

Tai, W.T., Chang, C.Y, Ing, C.H., and Chang,C.F. 2004. "Adsorption of acid dyes from aqueous 

solutions on activated bleaching earth ", J. Colloid and Interface Science, vol. 275, pp 72-78. 

Mon, J., Flury, M., and Harsh, J.B. 2005 . "Sorption of four triarylmethane dyes in a sandy soil 

determined by batch and column experiments ", Geoderma. in press. I 

1 

Mon, J., Flury . M., and Harsh. 2005 "A quantitative structure-activity relationship 

analysis of triarylmethane dye tracers ", Journal of Hydrology, in press. , 

I 

'OSAR) 
Characterization of Physical and Chemical Properties Submitted to the Agency in Support of 

Aquashade (23.6% and 2.39% Acid Yellow 23: 

Ellison. F.E. 1994. "Aqua Shade Blue 9'- Physical and Chemical Characteristics of Aqua Shade 

Blue 9: Color, Physical State, Odor, Boiling Point, Specific Gravity, Solubility, Vapor Pressure, 

pH, and Stability". Performed by Case Consulting Laboratories, Inc. Whippany, NJ and 

sponsored by Applied Biochemistry, Inc., Milwaukee, WI. Report Dated September 12, 1994. 

MRID 43503401 

I Ellison. F.E. 1994. "Aqua Shade Yellow 23"- Physical and Chemical Characteristics of Aqua 

shade Blue 9: Color, Physical State, Odor, Boiling Point, Specific Gravity, Solubility, Vapor 

Pressure, pH, and Stability". Performed by Case Consulting Laboratories, Inc. Whippany, NJ and I sp'onsored by Applied Biochemistry, Inc., Milwaukee, WI. Report Dated September 12, 1994. 

MRID 43503402 

5. Data Generation 

Structure-activity Estimates: 

EPI (Estimation Programs Interface) suiteTM (formerly known as EPIWIN): 

htt1p://www.epa.govlopptintr/exposure/docs/episuite.htm 

SRC. 2004. Syracuse Research Corporation. Interactive Physical Properties (PHYSPROP) 

Database Demo. Search terms: FD&C Blue. No. 1, FD&C Red No. 40, FD&C Yellow No. 5. 

(November 28,2004) http://www.syrres.corn/esc/physdemo.htm 

E. U.S. Environmental Protection Agency Documents: 

"Robust Summaries & Test Plans: C.I. Acid Yellow 23 (FD&C Yellow 5)", Submitted ti the 

Agency by the International Association of Color Manufacturers on March 10,2004. 

http://www.epa.gov/chemrtWciacdylo/c 15 133tc.htm 

"Background Document for Identification and Listing of Deferred Dye and Pigment Wastes, U.S. 

Environmental Protection Agency, Office of Solid Waste, Washington, D.C., June 1999. 

US Environmental Protection Agency. 1998. Guidelines for Ecological Risk Assessment. Risk 

Ass'tssment Forum. EPN630lR-95/002F, April 1998 

U.S . EPA. 1993. U. S . Environmental Protection Agency. Wildlife Exposure Factors Handbook. 

Volume I of 11. EPN6OOlR-931187a. Office of Research and Development, 

20460. 

E. U.S. Environmental Protection Agency, Office of Pesticide Programs Documents: 

I 
Memorandum: "Review of Phase 4 List D Package for Aquashade" (EFGWB # 93-01 19, 

93-0120; Chemical # 110301 and 110302; Case # 819437 and 819438; DP Barcode 

Dl 84289 and Dl 84278). Dated February 4,1993. 

"Reassessment of the Exemption from the Requirement of a Tolerance for the FDACertified Color Additives FD&C Blue No. 1, FD&C Red No. 40, and FD&C Yellow No. 

5 (Tartrazine)". U.S. Environmental Protection Agency, Office of Pesticides Programs. 

December 2 1,2004 

D. Internet Resources 

Food and Drugs Administration: 

http://www.cfsan.fda.gov/-dmslopa-col2.html 

Color Index International: 

http://www.colour-index.org/ 

Additional information was also obtained from TOXNET. 

http://toxnet.nlm.nih.gov/ 
Appendix A- Data Gaps 

Environmental Fate 

All of the Subdivision N, Environmental Fate Data Requirements maybe waived because 

of very low risk potential. They were identified as 'Reserved in 1993 (Memorandum: 

"Review of Phase 4 List D Package for Aquashade"; EFGWB #93-0119,93-0120; 

Chemicals 1 1030 1 and 1 10302; Case # 8 19437 and 8 19438; DP Barcode Dl 84289 and 

Dl 84278(. Dated February 4, 1993), pending on the results of required ecological 

toxicity studies. 

There are two major uncertainties on the environmental fate behavior of Acid Blue 9 and 

Acid Yellow 23 : 

(1) Structure-activity estimates of physical, chemical, and environmental fate 

behavior are not reliable for salts of large anions, such as Acid Yellow 23 and 

Acid Blue 9. 

(2) The major route of transformation of dyes such as Acid Blue 9 and Acid Yellow 

23 is indirect photolysis. However, the chemical identity of the photoproducts of 

these two dyes are not known. Biotransfomation under anaerobic conditions may 

also contribute to the dissipation of the dyes in water-sediments. The chemical 

identity of the metabolites is not known. 

However, after the review of the ecological toxicity studies and the risk assessment, the 

environmental fate data requirements placed in 1993 as "Reserved' can be waived. The. 

Environmental Fate and Effects Division is not requiring any new environmental fate 

data for the two dyes individually or for any of the end-use products (Aquashade, 

Aquashade OA, Pond Care AlgaeBlocker, Admiral Liquid, and Admiral WSP). 

Ecological Toxicity 

b. Effects 

The only EFED ecological toxicity data available is for the Aquashade product 

(23.6% Acid Blue 9 and 2.36% Acid Yellow 23) as the test substance. There are no 

EFED ecological toxicity data for Admiral Liquid or Admiral WSP, but HED received a 

study that found a laboratory rat LD50 equal to or more than 5,000 mgkg. However, 

since organisms will be only exposed to either 1 or 2 ppm in the environment, this 

uncertainty is minor. 

No chronic studies have been required, so it is not known what would happen 

over an extended exposure period. No studies on aquatic plants have been required, 

because they are the targets of Aquashade and it is assumed that all sudmerged aquatic 

plants in the treated pond will be killed. No terrestrial plant studies ha+e been required, 

because there is little expected exposure. An exception would be if anj. of these products 
might be applied to water used for irrigation. If that occurred, exposure to terrestrial 

plants may occur. 

It is not known what would happen to plants on the margins of a treated pond or 

growing as emergent vegetation in the shallows of the pond if the technical product or the 

end-use product was applied to them. Some labels warn that directly applying the 

products to emerged or terrestrial plants may cause burning. 

Because of the mode of action (blocking light that enters bodies of water), the low 

acute toxicity of this pesticide, and the relatively limited area involved in treatment, it is 

believed that additional studies are not needed. 
Appendix B Environmental Fate 

No Subdivision N Environmental Fate studies were submitted, as these data requirements 

were waived in 1993. Therefore, no data from guideline studies are included in this 

Appendix. 

Although the Environmental Fate and Effects Division does not usually use open 

literature data in its assessments, published data on the chemistry and behavior of dyes in 

aquatic systems was used to supplement the environmental fate assessment. Most of the 

data comes from structurally related dyes. Acid Blue 9 and Acid Yellow 23 are also food, 

drugs, and cosmetic colorants regulated by the Food and Drugs Administration. 

The environmental fate behavior of dyes such as Acid Yellow 23 and Acid Blue 9 can be 

summarized as follows: 

The dyes are fully ionized in the environmentally significant pH range of 5 to 9. 

1. There no hydrolyzable groups in these two dyes and, therefore, abiotic hydrolysis 

is not an important degradation pathway in the environment. 

2. Direct photolysis is slow, as indicated by the fading of the dyes in homogeneous 

aqueous media exposed to sunlight. However, indirect photolysis has been 

identified as the major route of degradation in environmental media. 

3. The dyes are resistant to biodegradation under aerobic conditions. Structural 

related dyes are difficult to biodegrade in activated sludge. However, redox 

reactions under anaerobic conditions appear to degrade the dyqs, at least to some 

extent. 

4. The two dyes are highly hydrophilic and do not have the potential to 

' bioaccumulate in fish. 
Appendix C Aquatic Exposure and Modeling 

The end-use products containing Acid Blue 9 and Acid Yellow 23 are directly applied to 

a confined water body and at the dose specified in the labels to attain a concentration of 1 

ppm or 2 ppm depending on the target pest. Because there are no runoff or spray drift 

components, the use of the simulation models GENEEC or PRZM and EXAMS is not 

appropriate. 

Aquatic exposure was based on the target concentration specified in the product labels, 

depending on the weed to be controlled. The maximum target concentration is 2 ppm (2 

ma) which is the concentration used to estimate risk for both aquatic and terrestrial 

animals 
Appendix D. Ecological Toxicity and Uncertainty in Ecological Toxicity Data I 

EPED requires the submission of six studies for all chemicals that are submitted 

for registration. Which additional data that is required depends upon the circumstances 

of the chemical and its use pattern. Birds are used as surrogates for terrestrial phase . 

amphibians and reptiles. Freshwater fish are used as surrogates for aquatic phase 

amphibians. 

The end-use products are not applied to a terrestrial environment. However, 

terrestrial animals (e.g., mammals and birds) are exposed to the dyes when they use 

treated water as the source of drinking water. 

In addition to data that was submitted by the registrants, EFED also reviewed 

studies from the open literature. These studies were obtained through the ORD Middle 

I Ecological Division's ECOTOX literature search and retrieval program managed in 

Duluth, Minnesota (http://www.epa.gov/ecotox). These data are consistent with the 

I contention that the dyes found in Aquashade and the other end-use products are 

practically nontoxic to the organisms tested. 

Bobwhite quail Acute Avian Oral 433367-01 LD~o & NOAEL z 2250 x 0.13 292.5 mglkg 

Mallard duck Acute Avian Oral 433367-02 LD50 & NOAEL z 2250 x 0.13 r 292.5 mglkg 

Bobwhite quail Avian Dietary 435034-03 LC50 & NOAEL5 2 620 x 0.13 2 80.6 ppm 

Mallard duck Avian Dietary 435034-04 ' LC50 & NOAELS 2 620 x 0.13 r 80.6 ppm 

Bluegill sunfish Acute Toxicity 432975-02 LC50 & NOAEC r 96 x 0.13 2 12.48 mg/L 

I 

Rainbow trout Acute Toxicity 432975-01 LC50 & NOAEC t 96 x 0.13 t 12.48 mgLL 

Daphnia magna Acute Toxicity 432975-03 I 

LC50 &NOAEC 2 97 x 0.13 2 12.61 mg& I 

1 All original tests were performed with the Typical End-use Product, Aquashade. No testing was performed on birds, fish and 

invertebrates with the end-use products Admiral Liquid and Admiral WSP, which have a higher percentage of the dyes than the 

tested Aquashade product. 

In addition to the uncertainty about the possible percent purity adjustment, Admiral WSP 

(49.27% acid blue 9 and 3.27% acid yellow 23) has about 50% greater concentration of 

dyes than Aquashade. To take this higher concentration into account, each toxicity 

endpoint could be multiplied by 2 resulting in an extrapolated avian acute N,OAEL 2 146 

mg/kg and aquatic organism NOAEC 26.3 mg/L. 
71-la 1433367-01 

Acute oral bobwhite 

quail ' 

71-la / 433367-02 

Acute oral mallard 

duck ' 

The formulated product (23.6% acid blue and LDS0 2 2250 mg eup /kg 

2.39% acid yellow dyes) was used. The study 

concentration was not adjusted for the purity of NOAEC = 

the active ingredients (the two dyes). The 2250 mg eup /kg 

results are milligrams of the end-use product. 

Five experimental doses were used - 292,486, 

810, 1350,2250 mg/L. There were no 

mortalities or other adverse effects. 

71-2b 1 435034-03 

Acute dietary 

bobwhite quail ' 

7 1-2a / 435034-04 

Acute dietary 

waterfowlmallard duck ' 

- - 

The formulated product (23.6% acid blue and LC5,, eup 2 5,620 ppm 

2.39% acid yellow dyes) was used. The study 

concentration was not adjusted for the purity of NOAEC eup = 

the active ingredients, (the two dyes). The 5,620 ppm eup 

results are parts per million of the end-use 

product. Five experirinental doses were used - 

562, 1000, 1780,316b, and 5620 ppm. There 

were no mortalities or other adverse effects. 

71-3 / 452811-01 The study used Admiral WSP, a purple powder 

Acute Wild Mammal formulation. The dye,s were not otherwise 

Toxicity using the identified. A group of 7-10 week old rats 

Laboratory rat (51sex) received a single dose of 5000 mgkg 

by gavage and were observed for 14 days. 

There were no mortalities, therefore the 

chemical was categoired as 'Toxicity 

Category IV." 

- -- 

Oral LDSO aa o 

> 5,000 mg eup /kg 

Oral LD50 o' & 0 

s2,500 mg a.i. /kg 

83-4 I 434109-01 Three generations of Long-Evans rats were NOAEL.&e = 

Reproduction study given tartrazine (92% a.i.) at 7.5,75, 225, and 750 mglkglday 

using the laboratory 750 mglkglday in their diet. This is consistent 

rat with no effects and addresses whether acid 

yellow 23 causes reproductive toxicity. There 

were no adverse treatment related effects. 

72-lc I 432975-01 The formulated product (23.6% acid blue LC5o eup 2 96 ma 

Acute coldwater fish- and 2.39% acid yellow dyes) was placed in NOAEC eup = 

Rainbow trout ' the water of aquaria. The study 96 mg/L 

72-la 1 432975-02 concentration was not adjusted for the 

Acute warmwater purity of the active ingredients (the two 

fish- bluegill dyes). The results are milligrams of the 

end-use product. ThB only dose used was sunfish ' I 

1 

96 ma. I 
72-2a / 432975-03 The formulated product (23.6% acid blue LC5() eup 2 97 mg/L 

Freshwater and 2.39% acid yellow dyes) was placed in NOAEC eup = 

invertebrate Daphnia the water of aquaria. The study 1 97 mg/L 

magna concentration was not adjusted for the 

purity of the active ingredients (the two 

dyes). The results are milligrams of the 

end-use product. The only dose used was 

97 mg/L. 

72- 1 a Freshwater cladocerans (Ceriodaphnia ECS0 = 5707 mg/L 

Warne & Schifko, dubia) were exposed to a dye for two days. 

1999 The EC50 put the chemical in the category 

FW Cladoceran "practically nontoxic." 

72-3a Exposure to marine habitats not expected N/A 

EstuarineIMarine 

Acute Toxicity with 

Sheepshead Minnow, 

Oyster, Mysids , 

72-4a Exposure to marine habitats not expected N/A 

Freshwater Fish Early 

Life Stage Fathead 

minnow 

72-4b 

Chronic effects not expected based on mode of 

N/ A 

Freshwater 

Invertebrate Life action and lack of effects in acute tests. 

Cycle- Water flea 

123-la If the label is changed and the water is not used Reserved 

Tier I1 Terrestrial Plant for irrigation, exposure to terrestrial plants 

Seedling Emergence would be unlikely. 

(GF 871) 

123-lb If the label is changed and the water is not used Reserved 

Tier I1 Terrestrial Plant for irrigation, exposure to terrestrial plants 

Vegetative Vigor (GF would be unlikely. 

871) 

123-2 Aquatic plants are the targets. Therefore, 

Tier I1 Aquatic Plant testing is not required. Release from 

Studies treated ponds to other water bodies 

considered a very low probability. , 
141-1 Mode of action suggests low likelihood of NIA 

Honey Bee Acute toxicity. The method of application 

Contact Toxicity minimizes exposure to terrestrial insects. 

Nonguideline Crayfish were exposed to 3 levels of NOAEL = 15 mg/L 

Spencer, 1984 Aquashade for 5 days. The oxygen use was 

Crayfish effected. The study was categorized as 

"slightly toxic." 

Nonguideline Laboratory rats were fed 4 levels of yellow 5 NOAEL = 

Borzelleca and for 900 days. The study was categorized as 984 mg/kg/day 

Hallagan, 1988. "practically nontoxic." LOffiL = 

Chronic toxicity1 264 1 mg/kg/day 

carcinogenicity - - - --- 

Nonguideline House mice (Mus musculus) were given food 7354 mglkglday 

Borzelleca, et al., 1990 with 3 levels of dye in their diet for 720 days. 

Chronic toxicity1 The study was characterized as "practically 

carcinogenicity in nontoxic." 

mice - --- 

I The study was done with Aquashade. The doses were calculated with the end-use product. 

The percentages of the dyes making up liquid is not clear. 

The study was done with Admiral Lake Colorant WSP. The LDso is based on the dry weight 

of the end-use product. The composition of the powder was not determined, but it is usually 

49.62% blue dye and 3.05% yellow dye. The LDSO a.i. cannot be determined, but is probably 

50% of the stated >5,000 mglkg. 

 he study was done with FD&C yellow dye (acid yellow 23 or 5), which is not an end-use 

products. 

Uncertainty in toxicity information 

Most of the toxicity results used for risk assessment were based on effects testing 

done with Aquashade (23.6% acid blue 9 and 2.39% acid yellow 23) ekcept the acute 

mammal LDSo was done with Admiral WSP and the reproductive mambalian NOAEL 

was done with acid yellow. 

There is some uncertainty in this approach because 1) the percent of the dyes in 

this formulation is unclear, and 2) some formulations have a higher percent of each dye 

than Aquashade, and it is not clear if the doses were expressed in terms of end-use 

product or active ingredients. The dyes in Aquashade are reported as bking 13% pure. 

To demonstrate the degree of uncertainty, the toxicity values may be mbltiplied by 0.1 to 

I 

I 
take these percentages into account. Note that the risk assessment was conducted with 

actual toxicity test results, not with these adjusted toxicity values. 

Table F-1. Summary of acute toxicity endpoints multiplied by 13% to composite for the 

possibility of the tested materials being only 13% pure. 

species Study T~P~' Source Extrapolated Endpoint 

Bobwhite quail Acute Avian Oral 433367-0 1 LD & NOAEL > 2250 x 0.13 2 292.5 mg&g 

so 

Mallard duck Acute Avia o Oral 433367-02 LD & NOAEL 2 2250 x 0.13 2 292.5 mgkg 50 

Bobwhlte qua11 Avian Dietary 435034-03 LC & NOAEL 2 620 x 0.13 2 80 6 ppm 50 

Mallard duck Avian Dietary 435034-04 LC & NOAEL 2 620 x 0.13 2 80.6 ppm 50 

Bluegill sunfish Acute Toxlcity 432975-02 LC &NOAEC >96x0.13 >1248mgn 50 

Rainbow trout Acute Toxicity 432975-01 LC & NOAEC 2 96 x 0.13 2 12 48 mgL 50 

Daphnra magna Acute Toxicity 432975-03 LC &NOAEC 297~0.13 >1261mgL 

50 

AH ofiginal tests Were performed with the typical end-use product, Aquashade. No testing was performed on bnds, fish and invertebrates 

with the end-use products Admiral Liquid and Admiral WSP, which have a higher percentage of the dyes bm the tested Aquashade product. 

In addition to the uncertainty about the possible percent purity adjustment, 

Admiral WSP (49.27% acid blue 9 and 3.27% acid yellow 23) has about 50% greater 

concentration of dyes than Aquashade. To take this higher concentration into account, 

each toxicity endpoint could be multiplied by 2 resulting in an extrapolated avian acute 

NOAEL 2 146 mglkg and aquatic organism NOAEC 2 6.3 mgL. 
Appendix E Status of Data Requirements 

1. Environmental Fate 

Subdivision N, Environmental Fate Data Requirements- Status of Data Requirements 

11 161-1 [Abiotic] Hydrolysis Reserved May be Waived No I I I 

Data Requirement 

162- 1 Aerobic Soil Metabolism Not Required Not Required No 

Status in 1993 

161 -2 [Direct] Photolysis in Water 

161-3 Photolysis on Soil 

11 162-2 Anaerobic Aquatic Metabolism I Not Required I Not Required I No 

Status after the Risk 

Assessment for 

Reregistration 

Reserved 

Not Required 

Additional 

Data 

Required 

162-3 Anaerobic Aquatic Metabolism 

11 164-1 Terrestrial Field Dissipation I Not ~e~uired Not Required No I I I 

May be Waived 

Not Required 

162-4 Aerobic Aquatic Metabolism 

163-1 Mobility in SoilsISediments 

11 164-2 Aquatic Field Dissipation Reserved May be Waived No 

No 

No 

I I 

Reserved 

Reserved 

Reserved 

Acute Avian Oral Quail TEP Y 433367-01 9-7-94 Acceptable 

7 1-1 (b) Acute Avian Oral Duck TEP Y 433367-02 9-7-94 Acceptable 

7 1-2(b) Avian DietaryfQuail TEP Y 435034-03 4-26-95 Acceptable 

7 1-2(b) Avian DietaryDuck TEP Y 435034-04 4-26-95 Acceptable 

71-3 Wild Mammal Toxicitya Y 4528 11-01 12-5-00 Acceptable 

7 1-4(a) Avian ReproductiveIQuail Waived 

7 1-4(b) Avian ReproductivetDuck Waived 

72-l(b) Acute Toxicity Bluegill TEP Y 432975-02 4-25-95 Acceptable 

72-l(d) Acute Toxicity Rainbow Trout TEP Y 432975-01 4-25-95 Acceptable 

- ---- 

May be Waived 

165-4 Bioaccumulation in Fish 

-- 

No 

May be Waived 

May be Waived 

No 

No 

I 

Reserved May be Waived 

The Log Kow does not 

trigger this data 

requirement No 
- This study was performed with Admiral WSP 

No additional effects data are being required. 
Appendix F Environmental Fate Bibliography 

Books 

Lynch, D.G. "Estimating the Properties of Synthetic Organic Dyes", in Handbook of 

Property Estimation Methods for Chemicals- Environmental Health Sciences, 

Edited by Robert S. Boethling and Donald Mackay. Published by Lewis Publishers, 

Boca Raton, FL; Pages 447- 467. And pertinent references therein. 

Marmion, D,M. Handbook of U.S. Colorants- Food, Drugs, and Medical Devices, 

Third Edition., 1991. Published by John Wiley and Sons, New York 

Helz, G.R., Zepp, R.G., and Crosby, D.G, Editors. 1994. Aquatic and surface 

photochemistry. Lewis Publishers, Boca Raton, Florida. 

~ourn'al Articles 

Jank. M., Koser, H., Liicking, F., Martienssen, M., and Wittchen, S. 1998, 

"Decolorization and Degradation of Erioglaucine (Acid Blue 9) Dye in Wastewater," 

Environmental Technology, v. 19(7), pp.74 1-747. 

Weber, E.J. and Adams, R.L. 1995. "Chemical and Sediment-Mediated Reduction of the 

Azo Dye Disperse Blue 79, " Environ. Sci. Technol. v.29, pp. 1163-1 170. 

I Baughman, G.L. 1995. "Fate of azo dyes in aquatic systems. Part 3: The role of 

suspended sediments in adsorption and reaction of acid and direct dyes," Dyes and 

Pigments, v.27, pp. 197-210. 

Brown, D. and Laboureur, P. 1983, "The Degradation of Dyestuffs: Part I- Primary 

biodegradation under anaerobic conditions, " Chemosphere, v. 122, pp. 397-404. 

Baran,W., Makowski, A., Wardas, W. 2003. "The influence of FeCl3 on the 

photocatalytic degradation of dissolved azo dyes in aqueous Ti02 suspensions, " 

Chemosphere, v.53, pp 82-95. 

Tai, W.T., Chang, C.Y, Ing, C.H., and Chang,C.F. 2004. "Adsorption of acid dyes from 

aqueous solutions on activated bleaching earth, " J. Colloid and Interface Science, vol. 

275, pp 72-78. 

Mon, J., Flury, M., and Harsh, J.B. 2005 . "Sorption of four triarylmethane dyes in a 

sandy soil determined by batch and column experiments, " Geoderrna. in press. I 

I 

Mon, J., Flury. M., and Harsh. 2005 "A quantitative structure-activity relationship 

(OSAR) analysis of triarylmethane dye tracers," Journal of Hydrology, in press. 

\ 
Appendix G Ecological Toxicity Bibliography 
Appendix H. Endangered and Threatened Species 

EFED also assessed the potential for Aquashade and other end-use products containing 

the Acid Yellow 23 and Acid Blue 9 dyes to potentially cause harm to endangered 

species. 

Terrestrial Animals 

Listed endangered or threatened species of birds, mammals and reptiles are not I 

I expected to be affected directly because exposure does not exceed the endangered species 

LOC. However, terrestrial animals may be affected indirectly if aquatic plants that are 

necessary for their survival are eliminated. The degree to which this might occur 

depends on the extent the treated water bodies are a critical component of their food 

supply. 

Terrestrial Plants 

Terrestrial plants have the potential for exposure if water treated with Aquashade 

or the other dye containing end-use products are used for irrigation. If irrigation is 

precluded from use on the labels, EFED concludes there is virtually no potential for 

exposure to terrestrial plants. 

Aquatic Animals 

Listed species of fish, invertebrates or amphibians are not expected to be affected 

directly because the exposure does not exceed the endangered species LOC. However, 

aquatic animals dwelling in treated ponds might have the potential for harm if the plants 

on which they depend for food and habitat are eliminated. The likelihood of this 

occurring depends on if any listed aquatic animal species would occur ,in these highly 

managed, often manmade, normally relatively small water bodies with usually no 

outflow, or if any, a very small amount. 

Aquatic Plants 

Listed species of aquatic plants which begin their life stages as submerged might 

be harmed if water bodies in which they are growing are treated while they are 

submerged. 

EFED has not fully analyzed the potential for endangered specits to be harmed 

through use of Aquashade and other end-use products. Such analysis a/ould focus on the 

overlap of specific species with potentially treated water bodies, and inlthe case of 

indirect effects, the likelihood that the treated water body comprises enbugh of a 

component of the listed species food or habitat base to actually impact the listed species, 

if that food or habitat was lost. 
Appendix K. Risk Quotient Calculation and Interpretation 

Risk characterization integrates the results of the exposure and ecotoxicity data to 

evaluate the likelihood of adverse ecological effects. The means of this integration is 

called the quotient method. Risk quotients (RQs) are calculated by dividing exposure 

estimates by acute and chronic ecotoxicity value and, in this case, it is a deterministic 

approach. 

RQ = EXPOSURE/TOXICITY 

RQs are then compared to OPP's levels of concern (LOCs). These LOCs are 

used by OPP to analyze potential risk to nontarget organisms and the need to consider 

regulatory action. The criteria indicate that a pesticide used as directed has the potential 

to cause adverse effects on nontarget organisms. LOCs currently address the following 

risk presumption categories: (1) acute -- potential for acute risk; regulatory action may be 

warranted in addition to restricted use classification, (2) acute restricted use -- the 

potential for acute risk, but may be mitigated through restricted use classification, (3) 

acute endangered species - endangered species may be adversely affected, and (4) 

chronic risk - the potential for chronic risk; regulatory action may be warranted. 

Currently, EFED does not perform assessments for chronic risk to plants, acute or 

chronic risks to nontarget insects, or chronic risk from granularlbait formulations to birds 

or mammals. 

The ecotoxicity test values (measurement endpoints) used in the acute and chronic 

risk quotients are derived from required studies. Examples of ecotoxicity values derived 

from short-term laboratory studies that assess acute effects are: (1) LCso (fish and birds), 

(2) LDSo (birds and mammals), (3) ECso (aquatic plants and aquatic invertebrates) and (4) 

EC25 (terrestrial plants). Examples of toxicity test effect levels derived from the results 

of long-term laboratory studies that assess chronic effects are: (1) LOAEC (birds, fish, 

and aquatic invertebrates), and (2) NOAEC (birds, fish and aquatic invertebrates). For 

birds and mammals, the NOAEC generally is used as the ecotoxicity test value in 

assessing chronic effects, although other values may be used when justified. However, 

the NOAEC is used if the measurement end point is production of offspring or survival 
Risk presumptions and the corresponding RQs and LOCs, are tabulated below. 

Table K-1: Risk presumptions for terrestrial animals 

Risk Presumption RQ LOC 

Birds 

Acute High Risk EEC'/LCSO or ~~50ls~ft~ or ~~50lda~' 0.5 

Acute Restricted Use EECILCSO or LDSOlsqft or LD5OIday (or LD50 < 50 mglkg) 0.2 

Acute Endangered Species EECILCSO or LDSOlsqft or LDSOIday 0.1 

Chronic Risk EECINOAEC 1 I 

Wild Mammals 

I 

Acute High Risk EECILCSO or LD5OIsqft or LD5OIday 0.5 

Acute Restricted Use EECILCSO or LD501sqft or LD5OIday (or LD50 < 50 mglkg) 0.2 

Acute Endangered Species EECILCSO or LDSOlsqft or LD5OIday 0.1 

Chronic Risk EECINOAEC 1 

1 abbreviation for Estimated Environmental Concentration (ppm) on avianlmammalian 

food items 

2 mg/ft2 

mg of toxicant consumed/day LD50 * wt. of a bird 
Table K-2. Risk Presumptions for Aquatic Animals 

Risk Presumption RQ LOC 

Acute Risk EEC'LCSO or EC50 0.5 

Acute Restricted Use EECLCSO or EC50 0.1 

Acute Endangered Species EECLC5O or EC50 0.05 

Chronic Risk EECIMATC or NOAEC 1 

1 EEC = (ppm or ppb) in water 

Table K-3. Risk presumptions for plants 

Risk Presumption RQ LOC 

Terrestrial and Semi-Aquatic Plants 

Acute Risk EEC'/EC~~ 1 

Acute High Risk EEC'/ECSO 1 1 

Acute Endangered Species EECECO5 or NOAEC 1 

1 

2 EEC = lbs ai1A 

EEC = (ppblppm) in water