ECOLOGICAL RISK ASSESSMENT

**Re-registration**

"Acid Blue 9\"(Erioglaucine) 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]{.underline}:     | [Acid Blue 9 (trisodium           |
|                                   | salt)]{.underline}                |
| PC Code Number:110302             |                                   |
|                                   | PC Code Number: 110301            |
| CAS Registry No.:1934-21-0        |                                   |
|                                   | CAS Registry No.:                 |
|                                   |                                   |
|                                   | Trisodium salt: 2650-18-2         |
|                                   |                                   |
|                                   | Triammonium 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                 |
+-----------------------------------+-----------------------------------+
| ![](media/image1.png)             | ![](media/image2.png)             |
+-----------------------------------+-----------------------------------+

Reviewers:

Ecological Effects: James Goodyear, Biologist

Environmental Fate: Silvia C. Termes, Chemist

[Secondary Reviewers]{.underline}:

Mark Corbin

Stephanie Syslo

Brian Anderson

[Branch Chief]{.underline}:

Daniel Rieder

Ecological Risk Branch III

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

II\. 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

III. Analysis.....................................................................19

A. Use Characterization

B. Exposure Characterization

C. Ecological Effects Characterization

IV\. Risk
Characterization......................................................32

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

Appendices

Appendix A Data Gaps

Appendix B Environmental Fate

Appendix C Aquatic Exposure and Modeling

Appendix D Ecological Toxicity and Uncertainty in Ecological Toxicity
Data

Appendix E Status of Data Requirements

Appendix F Environmental Fate Bibliography

Appendix G Ecological Toxicity Bibliography

Appendix H Endangered and Threatened Species

Appendix K Risk Quotient Calculation and Interpretation

Appendix L Papers accepted for ECOTOX, but not used in science chapter

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 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 cosmetics[^1].

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 described 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 [^2]

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 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, depending 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 Suite/EPIWIN[^3] 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 proposed, the need for new data
will be reevaluated.

*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 unlikely to volatilize from soils. Acid Blue 9 and Acid
Yellow 23 do not have the potential to bioaccumulate in fish.

D. Conclusions- Effects Characterization

Aquashade is a formulation that contains, as its active ingredients, a
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 toxicological, therefore little
toxicity is expected. The toxicity information available to characterize
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 studies 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 Development's (ORD) Middle Ecological Division in
Duluth through their literature searched 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 found that tested the dyes or
formulations on aquatic or terrestrial plants. In as much as this is a
control agent for submerged aquatic vegetation and algae, it is assumed
to have an adverse 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
terrestrial plants, so toxicity to this group of organisms is not
considered necessary. If treated waters are used for irrigation,
exposure to terrestrial plants may be possible and toxicity testing for
terrestrial plants would 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
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.

Indirect photolysis has been identified as the major route of
dissipation of the dyes in aquatic systems. However, the specific nature
of transformation products for Acid Blue 9 and Acid Yellow 23 is not
known. Although no kinetics data are available to assess how fast each
dye photodegrades, the geographical location, season, and
nature/concentration of natural photosensitizers would control the rate
of photolysis. Therefore, an uncertainty exists on the chemical nature
and concentration of photoproducts. In addition, anaerobic
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.

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 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 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 stated 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 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".

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 LD~50~ 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 of chemical impurities in the dyes 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.

Because of the mode of action (blocking sunlight energy that enters
bodies of water) and the low acute toxicity of this pesticide, it is
believed that additional ecological toxicity studies are not needed
unless new uses, methods of applications or new products are proposed.

**II. 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 dyes[^4] Acid Blue 9 and Acid 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 natural 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% Acid Blue 9 and
3.27% Acid Yellow 23). Additional information on these end-use products
is presented within the "Use Characterization" section.

The dyes Acid Blue 9 and Acid Yellow 23 are known by several different
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
Chemical 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 document, the names
"Acid Blue 9\" and "Acid Yellow 23\" are used, as they are identified as
such in the product 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 triammonium salt. Further information on the chemical identity of
the dyes are included in Table 2. However, because it is the anion of
the blue dye what exerts herbicidal activity by blocking sunlight
energy, the identity of the counter cation is not relevant to the
exposure and risk assessment when used as herbicides.

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 1\", whereas the triammonium salt (Blue 4; erioglaucine) is a
colorant in drugs and cosmetics (D&C). Use on foods, drugs, and
cosmetics require batch[^5] certification prior to use. While the nature
of the counter cation is important for regulation under FDCA, under
FIFRA 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 algicide/ 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 \[Acid Blue 9\]; 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
International[^7] standard for identification of pigments and dyes. The
Color Index classifies dyes and pigments by their composition.
Individual pigment or dye are identified by a unique Color Index Generic
Name (C.I. Name) and a Color Index Constitution Number (C.I. Number),
although the C.I. Generic Name is most commonly used..

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,

Acid Blue 9:

Acid Yellow 23:

Table 1 Chemical Nomenclature and Structure of the Dyes

+---------------------+------------------------+----------------------+
|                     | Acid Yellow 23         | Acid Blue 9          |
+---------------------+------------------------+----------------------+
| Common Name as      | Tartrazine             | Erioglaucine         |
| Appearing in the    |                        | (diammonium salt)    |
| labels of the       |                        |                      |
| Admiral Products    |                        |                      |
+---------------------+------------------------+----------------------+
| Chemical Name(s)    | 4,5-Dihydro-5-oxo      | *N*-Ethyl            |
|                     | -1-(4-sulfophenyl)-4-( | -*N*-(4-\[(4-{ethyl\ |
|                     | (4-sulfophenyl)azo)-1H | [(3-sulfophenyl)meth |
|                     | -pyrazole-3-carboxylic | yl\]amino}phenyl)(2- |
|                     | acid trisodium salt    | sulfophenyl)met      |
|                     |                        | hylene\]-2,5-cyclohe |
|                     | 3-carboxy-5-hydrox     | xadien-l-ylidene)-3- |
|                     | y-1-p-sulfophenyl-4-p- | sulfo                |
|                     | sulfophenylazopyrazole | benzenemethanaminium |
|                     | trisodium salt         | hydroxide inner      |
|                     |                        | salt, **trisodium    |
|                     | 5-                     | salt**               |
|                     | hydroxy-1-(p-sulfophen |                      |
|                     | yl)-4-((p-sulfophenyl) | *N*-Ethyl            |
|                     | azo)                   | -*N*-(4-\[(4-{ethyl\ |
|                     | pyrazole-3-carboxylic  | [(3-sulfophenyl)meth |
|                     | acid trisodium salt    | yl\]amino}phenyl)(2- |
|                     |                        | sulfophenyl)met      |
|                     |                        | hylene\]-2,5-cyclohe |
|                     |                        | xadien-l-ylidene)-3- |
|                     |                        | sulfo                |
|                     |                        | benzenemethanaminium |
|                     |                        | hydroxide inner      |
|                     |                        | salt, **triammonium  |
|                     |                        | salt**               |
+---------------------+------------------------+----------------------+
| Molecular Structure | ![](media/image3.png)  | Only the structure   |
|                     |                        | of the disodium salt |
|                     |                        | is represented. The  |
|                     |                        | diammonium salt has  |
|                     |                        | ammonium instead of  |
|                     |                        | sodium as the        |
|                     |                        | counter cation. It   |
|                     |                        | is the organic anion |
|                     |                        | what is responsible  |
|                     |                        | for the color of the |
|                     |                        | dye.                 |
|                     |                        |                      |
|                     |                        | !                    |
|                     |                        | [](media/image4.png) |
+---------------------+------------------------+----------------------+
| Synonyms            | [FD & C Yellow No.     | [Sodium              |
|                     | 5:]{.underline}        | Salt]{.underline}:   |
|                     |                        |                      |
|                     | 21CFR74.705 (Foods)    | Brilliant Blue       |
|                     |                        |                      |
|                     | 21CFR74.1705 (Drugs)   | CI Food Blue 2       |
|                     |                        |                      |
|                     | 21CFR74.2705           | Erioglaucine         |
|                     | (Cosmetics)            | trisodium salt       |
|                     |                        |                      |
|                     | These FD&C dyes are of | CI Food Blue 2.      |
|                     | high purity and        |                      |
|                     | require batch          | CI Number 42090      |
|                     | certification analysis |                      |
|                     | when used in food,     | CAS Registry No.     |
|                     | drugs, or cosmetics.   | 2650-18-2            |
|                     |                        |                      |
|                     | CI Food Yellow 4       | [Triammonium         |
|                     |                        | salt]{.underline}    |
|                     | CI Number 15985        |                      |
|                     |                        | Erioglaucine         |
|                     | CAS Registry No.       | diammonium salt      |
|                     | 1934-21-0              |                      |
|                     |                        | Alphazurine          |
|                     |                        |                      |
|                     |                        | CI Blue 8            |
|                     |                        |                      |
|                     |                        | CI 42090             |
|                     |                        |                      |
|                     |                        | CAS Reg. No.         |
|                     |                        | 3844-54-9            |
|                     |                        |                      |
|                     |                        | [FD&C Blue           |
|                     |                        | 1]{.underline}       |
|                     |                        |                      |
|                     |                        | 21CFR74.101 (Foods)  |
|                     |                        |                      |
|                     |                        | 21CFR74.1101 (Drugs) |
|                     |                        |                      |
|                     |                        | 21CFR74.2101         |
|                     |                        | (Cosmetics)          |
|                     |                        |                      |
|                     |                        | [D&C Blue            |
|                     |                        | 4]{.underline}       |
|                     |                        |                      |
|                     |                        | 21CFR74.1104 (Drugs) |
|                     |                        |                      |
|                     |                        | 21CFR.74.2104        |
|                     |                        | (Cosmetics)          |
+---------------------+------------------------+----------------------+
| Chemical Family     | Azo (monoazo) dye;     | A                    |
|                     | Contains one azo       | minotriphenylmethane |
|                     | group, - N=N-)         | dye                  |
+---------------------+------------------------+----------------------+

Other uses of the dyes include;

[Acid Yellow 23]{.underline}- Dye for wool and silk; As indicator for
chlorine estimations in biochemistry

[Acid Blue 9]{.underline}- Biological stain; Textile dye; Wood stain;
Indicator

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

*Physical and Chemical Properties*

The most distinct characteristic of dyes is that they absorb energy[^8]
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 visible 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 does not absorb light (i.e.,
the reflected color). In an "acid dye", the chromophore is part of a
negative ion (anion)[^9] . Therefore, the color is independent of the
counter cation. Table 2 summarizes information on the light absorption
characteristics of each dye.

Table 2- Electronic Absorption Spectra of Acid Yellow 23 and Acid Blue 9
in Water

  ----------------------- ----------------------- -----------------------
  Electronic Absorption   Acid Yellow 23          Acid Blue 9
  Spectra                                         

  Wavelength of           420- 430 range, ~max~, 620- 639 range; ~max~,
  Absorption Maximum      425                     630
  (~max~, in nm) in                              
  water                                           

  Reflected Color         Yellow                  Blue
  ----------------------- ----------------------- -----------------------

The physical and chemical properties of Acid Yellow 23 and Acid Blue 9
are summarized in Table 3.

Table 3- Physical and Chemical Properties of Acid Yellow 23 and Acid
Blue 9 (Estimated); Intrinsic properties

+-----------------------+-----------------------+-----------------------+
| Information           | Acid Yellow 23        | Acid Blue 9           |
+-----------------------+-----------------------+-----------------------+
| Empirical Formula     | C~16~                 | C~37~H~34~N~2~Na~2~   |
|                       | H~9~N~4~Na~3~O~9~S~2~ | O~9~ S~3~ (disodium   |
|                       |                       | salt)                 |
|                       |                       |                       |
|                       |                       | C~3                   |
|                       |                       | 7~H~34~N~2~(NH~4~)~2~ |
|                       |                       | O~9~ S~3~             |
|                       |                       |                       |
|                       |                       | (diammonium salt)     |
+-----------------------+-----------------------+-----------------------+
| Molecular Weight      | 534.37 (Marmion)      | 792.84 (disodium      |
|                       |                       | salt) (Marmion)       |
|                       |                       |                       |
|                       |                       | 782.96 (diammonium    |
|                       |                       | salt)                 |
+-----------------------+-----------------------+-----------------------+
| Physical State, as    | Bright orange-yellow  | Reddish-violet powder |
| pure chemicals        | powder (Marmion)      |                       |
|                       |                       | (Marmion)             |
+-----------------------+-----------------------+-----------------------+
| Vapor pressure,at 25 | 7.43 x 10^-22^        | 2.97 x 10^-42^        |
| C, in mmHg            |                       |                       |
|                       | there is some         | there is some         |
|                       | uncertainty in these  | uncertainty in these  |
|                       | estimated values,     | estimated values,     |
|                       | however they do not   | however they do not   |
|                       | significantly impact  | significantly impact  |
|                       | the assessment as the | the assessment as the |
|                       | vapor pressure are    | vapor pressure are    |
|                       | very low              | very low              |
|                       |                       |                       |
|                       | EPI Suite, 2004       | EPI Suite, 2004       |
|                       |                       |                       |
|                       | 25.0 (Reported in     | 28.6 (Reported in     |
|                       | 43503402)             | 43503401)             |
+-----------------------+-----------------------+-----------------------+
| Solubility in Water,  | 1 x 10^6^; EPI Suite, | 1.45 mg/L; EPI Suite, |
| 25C in               | 2004                  | 2004                  |
|                       |                       |                       |
| mg/L                  | "Completely soluble"  | "Completely soluble"  |
|                       | (Reported in          | (Reported in          |
| Log *n*-Octanol/Water | 43503402)             | 43503401)             |
| Partition Coefficient |                       |                       |
|                       | -10.7                 | -1.50                 |
|                       |                       |                       |
|                       | EPI Suite, 2004       | EPI Suite, 2004       |
|                       |                       |                       |
|                       | and SRC               | and SRC               |
+-----------------------+-----------------------+-----------------------+

+-----------------------------------------------------------------------+
| [Data Source]{.underline}                                             |
|                                                                       |
| Marmion= **Handbook of U.S. Colorants- Food, Drugs**, **and Medical   |
| Devices**, Third Edition., 1991.                                      |
|                                                                       |
| EPI Suite, 2004= Tolerance Reassessment Document                      |
|                                                                       |
| Submitted studies= 40503401 (Acid Blue 9) and 40503402 (Acid Yellow   |
| 23); According to the studies, 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     |
| likely to be associated with volatile impurities.                     |
|                                                                       |
| 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);              |
+-----------------------------------------------------------------------+

[Dissociation Constant (pKa]{.underline}): Acid dyes are weak acids. The
pK~a~ 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 photolysis[^11] Direct photolysis would be only significant in
clear, shallow water. But dyes have varying susceptibility to fading
when exposed to sunlight. Light fastness[^12] 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. Biotransformation
under anaerobic conditions has as also been documented for other
structurally related dyes. Acid Blue 9 and Acid Yellow 23 do not react
chemically with 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) Suite^TM^ (formerly known as
EPIWIN)[^13] , it was considered that the estimates are not reliable
because EPIWIN is not suitable for handling salts, particularly of those
with 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.

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) 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 non-edible 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 surface 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 and/or 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, hyacinths, 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 non-target or target plants or prey on the animals
that consume aquatic plants.

Risk to aquatic animals is based on registrant submitted acute, dietary
laboratory studies with aquatic vertebrates (rainbow trout and bluegill
sunfish) and invertebrates (water fleas).

The taxa evaluated for ecological effects are presented in Table 4.

+------------------------------+---------------------------------------+
| Table 4. Taxa evaluated for  |                                       |
| ecological effects in        |                                       |
| screening level risk         |                                       |
| assessments.                 |                                       |
+------------------------------+---------------------------------------+
| Taxon                        | Surrogate Species Used in Risk        |
|                              | Assessment                            |
+------------------------------+---------------------------------------+
| Birds ^a^                    | Mallard duck (*Anas playtrhynchos*)   |
|                              |                                       |
|                              | Bobwhite quail (*Colinus              |
|                              | virginianus*)                         |
+------------------------------+---------------------------------------+
| Mammals                      | Laboratory rat (*Rattus norvegicus)*  |
+------------------------------+---------------------------------------+
| Freshwater fish ^b^          | Bluegill sunfish (*Leopomis           |
|                              | macrochirus*)                         |
|                              |                                       |
|                              | Rainbow trout (*Oncorhynchus          |
|                              | mykiss*))                             |
+------------------------------+---------------------------------------+
| Freshwater invertebrates     | Water flea (*Daphnia magna*)          |
+------------------------------+---------------------------------------+
| Estuarine/marine fish        | Not Required because estuarine        |
|                              | exposure is considered unlikely       |
+------------------------------+---------------------------------------+
| Estuarine/marine             | Not Required because estuarine        |
| invertebrates                | 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 EFED 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.

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 Effects*

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 direction and
boundaries in the risk assessment for addressing risk management issues
of concern.

+------------------------------+---------------------------------------+
| **Table 5. Summary of        |                                       |
| assessment and measurement   |                                       |
| endpoints.**                 |                                       |
+------------------------------+---------------------------------------+
| **Assessment Endpoint**      | **Measurement Endpoint**              |
+------------------------------+---------------------------------------+
| 1\. Abundance (*i.e.*,       | 1a. Bobwhite quail and mallard duck   |
| survival and growth and      | acute oral LD~50~.                    |
| reproduction) of individuals |                                       |
| and populations of birds.    | 1b. Bobwhite quail and mallard duck   |
|                              | subacute dietary LC~50~.              |
|                              |                                       |
|                              | 1c. Avian reproductive NOAEL (in this |
|                              | case, no avian chronic data are       |
|                              | available, low chronic toxicity is    |
|                              | inferred based on low chronic         |
|                              | toxicity to mammals)                  |
+------------------------------+---------------------------------------+
| 2\. Survival of individual   | 2a. Laboratory rat acute oral LD~50~  |
| mammals and reproductive     | and reproduction test NOAEL.          |
| effects.                     |                                       |
+------------------------------+---------------------------------------+
| 3\. Survival and             | 3a. Rainbow trout and bluegill        |
| reproduction of freshwater   | sunfish acute LC~50~.                 |
| fish and invertebrates       |                                       |
|                              | 3c. Water flea acute LC~50~.          |
|                              |                                       |
|                              | 3d. Fish and invertebrate chronic     |
|                              | toxicity NOAEL.                       |
+------------------------------+---------------------------------------+
| 4\. Survival and Growth of   | 4\. Algae and vascular plant EC50 and |
| Aquatic Plants               | NOAEL                                 |
+------------------------------+---------------------------------------+
| 5\. Survival and Growth of   | 5\. Terrestrial monocot and dicot     |
| Terrestrial Plants           | vegetative vigor and seedling         |
|                              | emergent EC25 and NOAEL               |
+------------------------------+---------------------------------------+
| LD~50~ = Lethal dose to 50%  |                                       |
| of the test population.      |                                       |
|                              |                                       |
| LC~50~ (EC~50~) = Lethal     |                                       |
| (effective) concentration to |                                       |
| 50% of the test population.  |                                       |
|                              |                                       |
| EC~25~ = 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.       |                                       |
+------------------------------+---------------------------------------+

D. 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.

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

*Environmental 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 71 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 two studies were not conducted with a high purity dye (50% for
Acid Blue 9 and 28% for Acid Yellow 23)). These two studies were
submitted in support of the Aquashade product of 23.6% Acid 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. Most of the environ mental 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 Suite/EPIWIN 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 surrogate 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 (LC~50~s and
EC~50~s, respectively). Chronic measures are the no observed adverse
effect level or NOAEL which is the highest test level at which
significant effects were not observed.

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 nor spray drift are routes of exposure. Therefore, simulation
models (GENEEC; PRZM and 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

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 (71-1b avian oral,
71-2b avian dietary, 72-1b and 72-1d 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 testing, 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 other 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 represent
chronic risks based on a mammalian chronic study with the Acid Yellow
dye 23.

c\. Measures of Ecosystem and Receptor Characteristics

Aquashade's and the labels of the other end-use products specifically
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.

Since it is never applied to terrestrial environments, exposure to
terrestrial animals assessment estimating residues on terrestrial
vegetation and invertebrates was inappropriate. Instead calculations of
the amount in water consumed by birds or mammals drinking from the
treated body of water were done to derive a dose of Aquashade to birds
and mammals in three size classes.

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.

**III. 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.

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

+-----------------+-----------------+-----------------+-----------------+
| Product         | EPA Reg. No     | Composition (by | Target pests    |
|                 |                 | weight)         |                 |
|                 | Registrant      |                 |                 |
|                 |                 | Type of         |                 |
|                 | Label Date      | Formulation     |                 |
+-----------------+-----------------+-----------------+-----------------+
| Pond Care Algae | 8709-6          | 2.36 % Acid     | Control of      |
| Blocker         |                 | Blue 9          | growth of many  |
|                 | Aquarium        |                 | algae and       |
|                 | P               | 0.24 % Acid     | underwater      |
|                 | harmaceuticals, | Yellow 23       | aquatic weeds   |
|                 | Inc.            |                 |                 |
|                 |                 | Liquid          |                 |
|                 | February 6,     | formulation     |                 |
|                 | 2004            |                 |                 |
+-----------------+-----------------+-----------------+-----------------+
| Aquashade OA    | 33068-2         | 2.36 % Acid     | Control of      |
|                 |                 | Blue 9          | growth of many  |
| Aquashade       | Aquashade ,     |                 | algae and       |
|                 | Inc.            | 0.24 % Acid     | underwater      |
|                 |                 | Yellow 23       | aquatic weeds   |
|                 | February 23,    |                 |                 |
|                 | 1993            | Liquid          | Control of      |
|                 |                 | formulation     | growth of many  |
|                 | 3306-1          |                 | algae and       |
|                 |                 | 23.63% Acid     | underwater      |
|                 | Aquashade, Inc. | Blue 9 2.39%    | aquatic weeds   |
|                 |                 | Acid Yellow 23  |                 |
|                 | 1981            |                 |                 |
|                 |                 | Liquid          |                 |
|                 |                 | formulation     |                 |
+-----------------+-----------------+-----------------+-----------------+
| Admiral Liquid  | 67064-2         | 15.31% Acid     | Control algae   |
|                 |                 | Blue 8          | and aquatic     |
|                 | Becker          |                 | vegetation and  |
|                 | Underwood       | 1.00% Acid      | color the water |
|                 |                 | Yellow 23       |                 |
|                 | March 14, 2000  |                 |                 |
|                 |                 | Liquid          |                 |
|                 |                 | formulation     |                 |
+-----------------+-----------------+-----------------+-----------------+
| Admiral WSP     | 67064-1         | 49.72% Acid     | Control algae   |
|                 |                 | Blue 9          | and aquatic     |
|                 | Becker          |                 | vegetation and  |
|                 | Underwood       | 3.27% Acid      | color the water |
|                 |                 | Yellow 23       |                 |
|                 | August 17, 2001 |                 |                 |
|                 |                 | Concentrated    |                 |
|                 |                 | blend in        |                 |
|                 |                 | pre-measured    |                 |
|                 |                 | water soluble   |                 |
|                 |                 | packages        |                 |
+-----------------+-----------------+-----------------+-----------------+

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.

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

+-----------------------+-----------------------+-----------------------+
| Product               | Use Sites             | Time of Application   |
+-----------------------+-----------------------+-----------------------+
| Pond Care Algae       | Ornamental Ponds and  | Not specified         |
| Blocker               | recreational          |                       |
|                       | man-made, closed      | "Reapply as needed"   |
|                       | system ponds (Golf    |                       |
|                       | course lakes);        |                       |
|                       |                       |                       |
|                       | Rearing lakes for     |                       |
|                       | non-edible fish;      |                       |
|                       |                       |                       |
|                       | Fountains containing  |                       |
|                       | fish.                 |                       |
+-----------------------+-----------------------+-----------------------+
| Aquashade OA          | Fountains             | Not specified         |
|                       |                       |                       |
| Aquashade             | Aquarium              | Recommends            |
|                       |                       | application before    |
|                       | Ornamental Ponds      | weed growing season.  |
|                       |                       |                       |
|                       | Re-circulated or      | May be applied while  |
|                       | artificial            | ice is still covering |
|                       | waterscapes           | the water body        |
|                       |                       |                       |
|                       | Ideal Settings:       |                       |
|                       |                       |                       |
|                       | Shopping malls        |                       |
|                       |                       |                       |
|                       | Executive office      |                       |
|                       | parks                 |                       |
|                       |                       |                       |
|                       | Botanical gardens     |                       |
|                       |                       |                       |
|                       | Recreational and      |                       |
|                       | amusement parks.      |                       |
|                       |                       |                       |
|                       | Natural and manmade   |                       |
|                       | contained ponds and   |                       |
|                       | lakes including       |                       |
|                       | ornamental,           |                       |
|                       | recreational, fish    |                       |
|                       | rearing and fish      |                       |
|                       | farming ponds, golf   |                       |
|                       | course ponds          |                       |
|                       |                       |                       |
|                       | 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.          |                       |
+-----------------------+-----------------------+-----------------------+
| Admiral Liquid        | Natural or manmade    | Prior to or early in  |
|                       | ponds, lakes,         | the weed growing      |
|                       | fountains, fish       | season for optimum    |
|                       | farms, fish           | results               |
|                       | hatcheries, golf      |                       |
|                       | courses and swimming  | May be applied while  |
|                       | ponds                 | ice is still covering |
|                       |                       | the water body        |
+-----------------------+-----------------------+-----------------------+
| Admiral WSP           | Natural or manmade    | Prior to or early in  |
|                       | ponds, lakes,         | the weed growing      |
|                       | fountains, fish       | season for optimum    |
|                       | farms, fish           | results               |
|                       | hatcheries, golf      |                       |
|                       | courses and swimming  | Reapply "as needed"   |
|                       | ponds                 |                       |
+-----------------------+-----------------------+-----------------------+

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 the water bodies. The resulting concentration of the products in
water is fixed to "1 or 2 ppm", depending on the target 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

+-----------------------------------+-----------------------------------+
| Product                           | Dose                              |
+-----------------------------------+-----------------------------------+
| Pond Care Algae Blocker           | [Ornamental ponds and             |
|                                   | fountains]{.underline} 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   |
+-----------------------------------+-----------------------------------+
| Aquashade OA                      | "Small water bodies"              |
|                                   | (unspecified)                     |
| Aquashade                         |                                   |
|                                   | 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,             |
|                                   | spyrogyra-Water milfoil.          |
|                                   |                                   |
|                                   | 2 ppm to prevent production of    |
|                                   | Hydrilla's tubers.                |
+-----------------------------------+-----------------------------------+
| Admiral Liquid                    | 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               |
+-----------------------------------+-----------------------------------+
| Admiral WSP                       | Same as Admiral Liquid            |
+-----------------------------------+-----------------------------------+

Most of the labels contain precautionary language and/or 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.

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

+-----------------------------------+-----------------------------------+
| Products                          | Precautionary Language Included   |
|                                   | in the Labels                     |
+-----------------------------------+-----------------------------------+
| Pond Care Algae Blocker           | Non-target plants (water lilies,  |
|                                   | hyacinths, cattails) may suffer   |
|                                   | contact burns if material is      |
|                                   | accidentally poured directly into |
|                                   | them                              |
|                                   |                                   |
|                                   | Desirable submerged plants may    |
|                                   | also be affected due to reduced   |
|                                   | levels of sunlight                |
+-----------------------------------+-----------------------------------+
| Aquashade OA                      | None                              |
|                                   |                                   |
| Aquashade                         | 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 may be  |
|                                   | used for human consumption        |
+-----------------------------------+-----------------------------------+
| Admiral WSP                       | Same as for Admiral Liquid        |
+-----------------------------------+-----------------------------------+

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 intended    |
|                                   | for edible fish                   |
+-----------------------------------+-----------------------------------+
| Aquashade OA                      | Desirable plants such as water    |
|                                   | lilies may suffer contact burn if |
| Aquashade                         | material is accidentally poured   |
|                                   | on them directly                  |
|                                   |                                   |
|                                   | Shoreline non-target plants       |
|                                   | (cattails, 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.                        |
+-----------------------------------+-----------------------------------+
| Admiral Liquid                    | Shoreline non-target plants       |
|                                   | (cattails; water lilies) may      |
|                                   | suffer contact burn 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       |
|                                   | non-target areas. Do not apply    |
|                                   | where runoff is likely to occur.  |
+-----------------------------------+-----------------------------------+
| Admiral WSP                       | None specified                    |
+-----------------------------------+-----------------------------------+

B. Exposure Characterization

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 studies 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). Further 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 families but
both contain sulfonate groups. Because none of these two dyes contain
hydrolyzable groups, abiotic hydrolysis is not a degradation pathway for
these two dyes.

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 environment[^16], indirect 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
photolytically stable than Acid Blue 9, as the latter fades faster
(Marmion, 1991). Photo-degradation 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 photoredox 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 Adams, 1995; Jank, et al., 1998; Baughman,
1995). Some of the products of the anaerobic transformation of azo dyes
are aromatic amines. Therefore, in the environment azo dyes can lose
their color via photoredox reactions and by anaerobic biodegradation.
Anaerobic biodegradation is most likely to occur in sediments.

Fading can also occur by reaction with trace metals, such as zinc, tin,
aluminum, iron, and copper, mostly via redox reactions. However, fading
is primarily a photochemical process.

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 predominantly associated with the water
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 \<\< 1, they are not likely to
bioaccumulate in fish.

2\. Measures of Aquatic Exposure

a\. Aquatic Exposure Modeling

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
performed.and it was concluded that there was no 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 ecological 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 Yellow 23
of uncertain purity. Therefore, an assessment based on each individual
dye was not possible. The ecological toxicity of products with a higher
percentage of each individual dye and/or different 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 100% purity of each dye (Source: Product
labels)

+-----------------+-----------------+-----------------+-----------------+
| Product         | Application     | Concentration   | Concentration   |
|                 | Rate of Product | of Acid Blue 9, | of Acid Yellow  |
|                 | as target       | in mg/L         | 23, in mg/L     |
|                 | concentration,  |                 |                 |
|                 | ppm; mg/L       |                 |                 |
+-----------------+-----------------+-----------------+-----------------+
| Aquashade OA    | 1               | 0.024           | 0.0024          |
|                 |                 |                 |                 |
| Aquashade       | 2               | 0.048           | 0.0048          |
|                 |                 |                 |                 |
|                 | 1               | 0.24            | 0.024           |
|                 |                 |                 |                 |
|                 | 2               | 0.48            | 0.048           |
+-----------------+-----------------+-----------------+-----------------+
| Pond Care       | 1               | 0.024           | 0.0023          |
| AlgaeBlocker    |                 |                 |                 |
+-----------------+-----------------+-----------------+-----------------+
| Admiral Liquid  | 1               | 0.15            | 0.01            |
|                 |                 |                 |                 |
|                 | 2               | 0.3             | 0.02            |
+-----------------+-----------------+-----------------+-----------------+
| Admiral WSP     | 1               | 0.497 (0.5)     | 0.033           |
|                 |                 |                 |                 |
|                 | 2               | 0.99            | 0.066           |
+-----------------+-----------------+-----------------+-----------------+

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 bodies[^17]. 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 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) × 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) 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 =  (body weight in kg) ^^, where  = 0.059 and  = 0.67**

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 =  (body weight in kg) ^^, where  = 0.099 and  = 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).

+-----------+--------------+--------------------------+--------------+
| **Table   |              |                          |              |
| 13.       |              |                          |              |
| Exposure  |              |                          |              |
| estimates |              |                          |              |
| for birds |              |                          |              |
| and       |              |                          |              |
| mammals   |              |                          |              |
| via       |              |                          |              |
| co        |              |                          |              |
| nsumption |              |                          |              |
| of water  |              |                          |              |
| con       |              |                          |              |
| taminated |              |                          |              |
| with      |              |                          |              |
| Aquashade |              |                          |              |
| at a      |              |                          |              |
| maximum   |              |                          |              |
| c         |              |                          |              |
| alculated |              |                          |              |
| conc      |              |                          |              |
| entration |              |                          |              |
| of 2      |              |                          |              |
| mg/L.**   |              |                          |              |
+-----------+--------------+--------------------------+--------------+
| **Taxon** | **Body**     | **Estimated Water**      | **Estimated  |
|           |              |                          | Exposure^a^  |
|           | **Weight     | **Consumption (L)**      | (mg)**       |
|           | (g)**        |                          |              |
+-----------+--------------+--------------------------+--------------+
| Birds     | 20           | 0.0043                   | 0.008        |
+-----------+--------------+--------------------------+--------------+
|           | 100          | 0.013                    | 0.026        |
+-----------+--------------+--------------------------+--------------+
|           | 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     |              |                          |              |
| co        |              |                          |              |
| nsumption |              |                          |              |
| (L) ×     |              |                          |              |
| Water     |              |                          |              |
| conc      |              |                          |              |
| entration |              |                          |              |
| 2 mg/L).  |              |                          |              |
+-----------+--------------+--------------------------+--------------+

b\. Residue Studies

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

C. Ecological Effects Characterization.

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 algicide/aquatic herbicide. These products
are applied directly 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 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 fulfill 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.

+------------+----------------------+----------+-----------------------+
| **Table    |                      |          |                       |
| 14.        |                      |          |                       |
| Summary of |                      |          |                       |
| most       |                      |          |                       |
| sensitive  |                      |          |                       |
| acute      |                      |          |                       |
| data. No   |                      |          |                       |
| chronic    |                      |          |                       |
| data were  |                      |          |                       |
| required   |                      |          |                       |
| for birds, |                      |          |                       |
| fish or    |                      |          |                       |
| aquatic    |                      |          |                       |
| invert     |                      |          |                       |
| ebrates.** |                      |          |                       |
+------------+----------------------+----------+-----------------------+
| *          | **Study Type^1^**    | **       | **Results^2^**        |
| *Species** |                      | Source** |                       |
+------------+----------------------+----------+-----------------------+
| Test       |                      |          |                       |
| Material   |                      |          |                       |
| Aquashade  |                      |          |                       |
+------------+----------------------+----------+-----------------------+
| Bobwhite   | Acute Avian Oral     | 4        | LD50 & acute NOAEC   |
| quail      |                      | 33367-01 | 2250 mg/kg            |
+------------+----------------------+----------+-----------------------+
| Mallard    | Acute Avian Oral     | 4        | LD50 & acute NOAEC   |
| duck       |                      | 33367-02 | 2250 mg/kg            |
+------------+----------------------+----------+-----------------------+
| Bobwhite   | Avian Dietary        | 4        | LC50 & acute          |
| quail      |                      | 35034-03 | NOAEC 5, 620ppm      |
+------------+----------------------+----------+-----------------------+
| Mallard    | Avian Dietary        | 4        | LC50 & acute NOAEC   |
| duck       |                      | 35034-04 | 5,620ppm              |
+------------+----------------------+----------+-----------------------+
| Bluegill   | Fish Toxicity        | 4        | LC50 & acute NOAEC   |
| sunfish    | Bluegill             | 32975-02 | 96 mg/L               |
+------------+----------------------+----------+-----------------------+
| Rainbow    | Fish Toxicity        | 4        | LC50 & acute NOAEC   |
| trout      | Rainbow Trout        | 32975-01 | 96 mg/L               |
+------------+----------------------+----------+-----------------------+
| *Daphnia   | Invertebrate         | 4        | LC50 & acute NOAEC   |
| magna*     | Toxicity             | 32975-03 | 97 mg/L               |
+------------+----------------------+----------+-----------------------+
| Test       |                      |          |                       |
| Material   |                      |          |                       |
| Admiral    |                      |          |                       |
| WSP        |                      |          |                       |
+------------+----------------------+----------+-----------------------+
| Laboratory | Wild Mammal Acute    | 4        | LD50  &   5,000    |
| rat        |                      | 52811-01 | mg/kg                 |
+------------+----------------------+----------+-----------------------+
| ^1^ All    |                      |          |                       |
| were       |                      |          |                       |
| performed  |                      |          |                       |
| with the   |                      |          |                       |
| Typical    |                      |          |                       |
| End-use    |                      |          |                       |
| Product,   |                      |          |                       |
| Aquashade  |                      |          |                       |
| or Admiral |                      |          |                       |
| WSP        |                      |          |                       |
|            |                      |          |                       |
| ^2^ The    |                      |          |                       |
| results    |                      |          |                       |
| refer to   |                      |          |                       |
| the        |                      |          |                       |
| con        |                      |          |                       |
| centration |                      |          |                       |
| of the     |                      |          |                       |
| end-use    |                      |          |                       |
| product.   |                      |          |                       |
+------------+----------------------+----------+-----------------------+

Birds and mammals are expected to drink from ponds that have been
treated with the end-use products. EFED calculated an estimated dose
that they would receive from that water (see Table 10).

1\. Terrestrial Effects

*Avian effects*

The avian oral acute toxicity studies for both the bobwhite quail and
the mallard duck found LD~50~\'s and NOAECs equal to or greater than
2,250 mg/kg. The avian dietary toxicity studies found LC~50~s 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 LD~50~ equal to
or greater than 5,000 mg/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 LD~50~
(2,250 mg/kg-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 LD~50~ value of 5,000 mg/kg-bw (MRID
452811-01) 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        |             |                   |                     |
| c           |             |                   |                     |
| ontaminated |             |                   |                     |
| water,      |             |                   |                     |
| expressed   |             |                   |                     |
| in terms of |             |                   |                     |
| a single    |             |                   |                     |
| Aquashade   |             |                   |                     |
| dose        |             |                   |                     |
| (mg).**     |             |                   |                     |
+-------------+-------------+-------------------+---------------------+
| Animals     | Body Weight | LD~50~            | Derived Median      |
|             | (kg)        |                   |                     |
|             |             | (mg/kg-bw)        | Lethal Dose (mg) a  |
+-------------+-------------+-------------------+---------------------+
| ~Birds~     | ~0.020~     | ~2,250~          | ~45~               |
+-------------+-------------+-------------------+---------------------+
|             | ~0.100~     | ~2,250~          | ~225~              |
+-------------+-------------+-------------------+---------------------+
|             | ~1.000~     | ~2,250~          | ~2,250~            |
+-------------+-------------+-------------------+---------------------+
| ~Mammals~   | ~0.015~     | ~5,000~          | ~75~               |
+-------------+-------------+-------------------+---------------------+
|             | ~0.035~     | ~5,000~          | ~175~              |
+-------------+-------------+-------------------+---------------------+
|             | ~1.000~     | ~5,000~          | ~5,000~            |
+-------------+-------------+-------------------+---------------------+
| ~**a**~     |             |                   |                     |
| Derived     |             |                   |                     |
| Median      |             |                   |                     |
| Lethal Dose |             |                   |                     |
| (mg) = body |             |                   |                     |
| weight (kg) |             |                   |                     |
| × LD~50~    |             |                   |                     |
| (mg/kg body |             |                   |                     |
| weight).    |             |                   |                     |
+-------------+-------------+-------------------+---------------------+

*Terrestrial Plant effects*

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. 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 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 formulations. 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 LC~50~s 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 magna*) study found that the LC~50~
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 adverse effects to organisms other
than those tested by the registrant. None of the data located through
ECOTOX were used in the assessment. See Appendix F for a summary table
of data located 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 concentration 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 end-use 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.

+--------+---------+--------------------+------------+---------------+
| *      |         |                    |            |               |
| *Table |         |                    |            |               |
| 16.    |         |                    |            |               |
| Acute  |         |                    |            |               |
| Risk   |         |                    |            |               |
| Quo    |         |                    |            |               |
| tients |         |                    |            |               |
| (RQ)   |         |                    |            |               |
| for    |         |                    |            |               |
| birds  |         |                    |            |               |
| and    |         |                    |            |               |
| m      |         |                    |            |               |
| ammals |         |                    |            |               |
| e      |         |                    |            |               |
| xposed |         |                    |            |               |
| to     |         |                    |            |               |
| Aqu    |         |                    |            |               |
| ashade |         |                    |            |               |
| t      |         |                    |            |               |
| hrough |         |                    |            |               |
| consu  |         |                    |            |               |
| mption |         |                    |            |               |
| of     |         |                    |            |               |
| contam |         |                    |            |               |
| inated |         |                    |            |               |
| wa     |         |                    |            |               |
| ter.** |         |                    |            |               |
+--------+---------+--------------------+------------+---------------+
| **     | **Body  | **Estimated        | **Derived  | **Acute RQ**  |
| Taxa** | weight  | Environmental      | Medium     |               |
|        | (g)**   | Exposure^a^ (mg)** | Lethal     | **EEC/DMDL**  |
|        |         |                    | Dose       |               |
|        |         |                    | (DMDL)b**  |               |
+--------+---------+--------------------+------------+---------------+
| Birds  | 20      |  0.01              |     45    | \< 0.01       |
+--------+---------+--------------------+------------+---------------+
|        | 100     |   0.02             |    225    | \< 0.01       |
+--------+---------+--------------------+------------+---------------+
|        | 1,000   |  0.12              |  2,250    | \< 0.01       |
+--------+---------+--------------------+------------+---------------+
| M      | 15      | \< 0.01            |     75    | \< 0.01       |
| ammals |         |                    |            |               |
+--------+---------+--------------------+------------+---------------+
|        | 35      |  \< 0.01           |    175    | \< 0.01       |
+--------+---------+--------------------+------------+---------------+
|        | 1,000   | \< 0.10            |  5,000    | \< 0.01       |
+--------+---------+--------------------+------------+---------------+
| *      |         |                    |            |               |
| *^a^** |         |                    |            |               |
| Est    |         |                    |            |               |
| imated |         |                    |            |               |
| E      |         |                    |            |               |
| nviron |         |                    |            |               |
| mental |         |                    |            |               |
| Ex     |         |                    |            |               |
| posure |         |                    |            |               |
| (EEC)  |         |                    |            |               |
| =      |         |                    |            |               |
| Daily  |         |                    |            |               |
| water  |         |                    |            |               |
| consu  |         |                    |            |               |
| mption |         |                    |            |               |
| (L) ×  |         |                    |            |               |
| Water  |         |                    |            |               |
| c      |         |                    |            |               |
| oncent |         |                    |            |               |
| ration |         |                    |            |               |
| (      |         |                    |            |               |
| mg/L). |         |                    |            |               |
|        |         |                    |            |               |
|   R    |         |                    |            |               |
| ounded |         |                    |            |               |
| to two |         |                    |            |               |
| d      |         |                    |            |               |
| ecimal |         |                    |            |               |
| p      |         |                    |            |               |
| laces. |         |                    |            |               |
|        |         |                    |            |               |
| *      |         |                    |            |               |
| *^b^** |         |                    |            |               |
| D      |         |                    |            |               |
| erived |         |                    |            |               |
| Median |         |                    |            |               |
| Lethal |         |                    |            |               |
| Dose   |         |                    |            |               |
| (mg) = |         |                    |            |               |
| body   |         |                    |            |               |
| weight |         |                    |            |               |
| (kg) × |         |                    |            |               |
| LD~50~ |         |                    |            |               |
| (mg/kg |         |                    |            |               |
| body   |         |                    |            |               |
| we     |         |                    |            |               |
| ight). |         |                    |            |               |
|        |         |                    |            |               |
| *      |         |                    |            |               |
| *^c^** |         |                    |            |               |
|  Acute |         |                    |            |               |
| RQ     |         |                    |            |               |
|  = Est |         |                    |            |               |
| imated |         |                    |            |               |
| E      |         |                    |            |               |
| nviron |         |                    |            |               |
| mental |         |                    |            |               |
| Ex     |         |                    |            |               |
| posure |         |                    |            |               |
| /      |         |                    |            |               |
| C      |         |                    |            |               |
| oncent |         |                    |            |               |
| ration |         |                    |            |               |
| D      |         |                    |            |               |
| erived |         |                    |            |               |
| 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*

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 direct 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 lb ai/acre.

Applying 1 lb ai/acre to short grass would result in residues no greater
than 240 ppm. For a small (15g) mammal that eats close to its body
weight this would still result in a dose per animal much less than the
lowest toxicity value for mammals. See appendix E for calculations.

2\. Non-target Aquatic Animals and Plants

Aquashade and any of the other end-use product containing Acid Blue 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
Aquashade, therefore, the highest EECs (2 mg/L; for Admiral Liquid and
Admiral WSP) and the lowest toxicity values were used. The concentration
in the treated systems will be higher than any body of water that
accidentally receives (by flooding, *etc.*) a dose of Aquashade. A
summary of aquatic acute RQs is presented in Table 17.

  ---------------- ---------- ------------- -------------------- -------------
  **Table                                                        
  17.  Estimated                                                 
  acute risk                                                     
  quotients (RQ)                                                 
  for aquatic                                                    
  animals exposed                                                
  to Aquashade. In                                               
  this screening                                                 
  analysis only                                                  
  the highest                                                    
  Estimated                                                      
  Environmental                                                  
  Concentration                                                  
  (EEC = 2 mg/L)                                                 
  and lowest                                                     
  NOAECs were                                                    
  used.**                                                        

  **Animal**       **EEC      **NOAEC**     **RQ (EEC / NOAEC)** **LOCs
                   (mg/L)**                                      Exceeded**

  Bluegill sunfish 2          96 mg/L       \<0.01               none

  Rainbow trout    2          96 mg/L       \<0.01               none

  *Daphnia magna*  2          97 mg/L       \<0.01               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.

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 affect 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 these 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 co-located 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 man-made 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 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 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 indirect 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 estimations (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, this 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. The physical and chemical properties
were submitted in support of Aquashade (23.6% Acid Blue 9 and 2.39 Acid
Yellow 2.39% registration In is unclear if these percentages take into
account the purity 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

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 (15.31% 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 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." Therefore the
exact composition of the toxicant that was used in the studies is not
certain. The use of the word "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**

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E.  Open Literature

[Books]{.underline}

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, 2000;
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.

[Journal Articles]{.underline}

Jank. M., Köser, H., Lücking, F., Martienssen, M., and Wittchen, S.
1998, "*Decolorization and Degradation of Erioglaucine (Acid Blue 9) Dye
in Wastewater"*, [Environmental Technology]{.underline}, v.19(7),
pp.741-747.

Weber, E.J. and Adams, R.L. 1995. *"Chemical and Sediment-Mediated
Reduction of the Azo* *Dye Disperse Blue 79\"*, [Environ. Sci.
Technol]{.underline}. v.29, pp. 1163-1170.

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 FeCl~3~ on
the photocatalytic degradation of dissolved azo dyes in aqueous TiO~2~
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.

Mon, J., Flury. M., and Harsh. 2005 *"A quantitative structure-activity
relationship (OSAR) analysis of triarylmethane dye tracers"*, Journal of
Hydrology, in press.

[Characterization of Physical and Chemical Properties Submitted to the
Agency in Support of Aquashade (23.6% and 2.39% Acid Yellow
23]{.underline}:

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**

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 sponsored by Applied Biochemistry, Inc., Milwaukee, WI. Report Dated
September 12, 1994.

**MRID 43503402**

5.  Data Generation

Structure-activity Estimates:

EPI (Estimation Programs Interface) Suite^TM^ (formerly known as
EPIWIN):

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)

E.  [U.S. Environmental Protection Agency Documents]{.underline}:

"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.

"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 Assessment Forum. EPA/630/R-95/002F, April 1998

U.S. EPA. 1993. U. S. Environmental Protection Agency. Wildlife Exposure
Factors Handbook. Volume I of II. EPA/600/R‑93/187a. Office of Research
and Development, Washington, D. C. 20460.

E.  [U.S. Environmental Protection Agency, Office of Pesticide Programs
    Documents:]{.underline}

Memorandum: "Review of Phase 4 List D Package for Aquashade" (EFGWB \#
93-0119, 93-0120; Chemical \# 110301 and 110302; Case \# 819437 and
819438; DP Barcode D184289 and D184278). Dated February 4, 1993.

"Reassessment of the Exemption from the Requirement of a Tolerance for
the FDA-Certified 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 21, 2004

D.  [Internet Resources]{.underline}

Food and Drugs Administration:

Color Index International:

Additional information was also obtained from TOXNET.

**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 110301 and 110302; Case
\# 819437 and 819438; DP Barcode D184289 and D184278(. 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.
    Biotransformation 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).

Number range CHAPTER*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 mg/kg. 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 submerged aquatic plants in the treated
    pond will be killed. No terrestrial plant studies have been
    required, because there is little expected exposure. An exception
    would be if any 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 dyes, 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 mg/L) which is the concentration used
to estimate risk for both aquatic and terrestrial animals

**Appendix D. Ecological Toxicity and Uncertainty in Ecological Toxicity
Data**

EFED 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 Ecological Division's ECOTOX literature search
and retrieval program managed in Duluth, Minnesota
(http://www.epa.gov/ecotox). These data are consistent with the
contention that the dyes found in Aquashade and the other end-use
products are practically nontoxic to the organisms tested.

  ---------------------------------------------------------------------------
  **Table D-1.                               
  Summary of most                            
  sensitive acute                            
  toxicity                                   
  endpoints                                  
  multiplied by                              
  13% based on                               
  percent purity                             
  of the dyes in                             
  aquashade.**                               
  --------------- ------------- ------------ --------------------------------
  Species         Study Type^1^ Source       Extrapolated Endpoint

  Bobwhite quail  Acute Avian   433367-01    LD~50~ & NOAEL  2250 x 0.13 
                  Oral                       292.5 mg/kg

  Mallard duck    Acute Avian   433367-02    LD~50~ & NOAEL  2250 x 0.13 
                  Oral                       292.5 mg/kg

  Bobwhite quail  Avian Dietary 435034-03    LC~50~ & NOAEL5  620 x 0.13 
                                             80.6 ppm

  Mallard duck    Avian Dietary 435034-04    LC~50~ & NOAEL5  620 x 0.13 
                                             80.6 ppm

  Bluegill        Acute         432975-02    LC~50~ & NOAEC  96 x 0.13 
  sunfish         Toxicity                   12.48 mg/L

  Rainbow trout   Acute         432975-01    LC~50~ & NOAEC  96 x 0.13 
                  Toxicity                   12.48 mg/L

  *Daphnia magna* Acute         432975-03    LC~50~ & NOAEC  97 x 0.13 
                  Toxicity                   12.61 mg/L

  ^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 NOAEL  146
mg/kg and aquatic organism NOAEC 6.3 mg/L.

+-----------------+---------------------------------+------------------+
| **Table         |                                 |                  |
| D               |                                 |                  |
| -2.  EcoEffects |                                 |                  |
| Data including  |                                 |                  |
| Data from Open  |                                 |                  |
| Literature**    |                                 |                  |
+=================+=================================+==================+
| **Guideline /** | **Discussion**                  | **Results**      |
|                 |                                 |                  |
| **MRID or       |                                 |                  |
| Citation**      |                                 |                  |
+-----------------+---------------------------------+------------------+
| 71-1a /         | The formulated product  (23.6%  | LD~50~  2250 mg |
| 433367-01       | acid blue and 2.39% acid yellow | eup /kg          |
|                 | dyes) was used. The study       |                  |
| Acute oral      | concentration was not adjusted  | NOAEC =          |
| bobwhite        | for the purity of the active    |                  |
| quail ^1^       | 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.          |                  |
+-----------------+---------------------------------+------------------+
| 71-1a /         |                                 |                  |
| 433367-02       |                                 |                  |
|                 |                                 |                  |
| Acute oral      |                                 |                  |
| mallard         |                                 |                  |
| duck ^1^        |                                 |                  |
+-----------------+---------------------------------+------------------+
| 71-2b /         | The formulated product  (23.6%  | LC~50~  eup     |
| 435034-03       | acid blue and 2.39% acid yellow | 5,620 ppm        |
|                 | dyes) was used. The study       |                  |
| Acute dietary   | concentration was not adjusted  | NOAEC  eup =     |
| bobwhite        | for the purity of the active    | 5,620 ppm eup    |
| quail ^1^       | ingredients (the two dyes). The |                  |
|                 | results are parts per million   |                  |
|                 | of the end-use product. Five    |                  |
|                 | experimental doses were used -  |                  |
|                 | 562, 1000, 1780, 3160, and      |                  |
|                 | 5620 ppm. There were no         |                  |
|                 | mortalities or other adverse    |                  |
|                 | effects.                        |                  |
+-----------------+---------------------------------+------------------+
| 71-2a /         |                                 |                  |
| 435034-04       |                                 |                  |
|                 |                                 |                  |
| Acute dietary   |                                 |                  |
| waterfowl-      |                                 |                  |
|                 |                                 |                  |
| mallard         |                                 |                  |
| duck ^1^        |                                 |                  |
+-----------------+---------------------------------+------------------+
| 71-3 /          | The study used Admiral WSP, a   | Oral LD~50~      |
| 452811-01       | purple powder formulation. The  | ~ & ~          |
|                 | dyes were not otherwise         |                  |
| Acute Wild      | identified. A group of 7-10     | \> 5,000 mg eup  |
| Mammal Toxicity | week old rats (5/sex) received  | /kg              |
| using the       | a single dose of 5000 mg/kg by  |                  |
| Laboratory      | gavage and were observed for 14 | Oral LD~50~      |
| rat ^2^         | days. There were no             | ~ & ~          |
|                 | mortalities, therefore the      |                  |
|                 | chemical was categorized as     | 2,500 mg a.i.   |
|                 | "Toxicity Category IV."         | /kg              |
+-----------------+---------------------------------+------------------+
| 83-4 /          | Three generations of Long-Evans | NOAEL ~ & ~ =  |
| 434109-01       | rats were given tartrazine (92% |                  |
| Reproduction    | a.i.) at 7.5, 75, 225, and 750  | 750 mg/kg/day    |
| study using the | mg/kg/day in their diet. This   |                  |
| laboratory      | is consistent with no effects   |                  |
| rat ^3^         | and addresses whether acid      |                  |
|                 | yellow 23 causes reproductive   |                  |
|                 | toxicity. There were no adverse |                  |
|                 | treatment related effects.      |                  |
+-----------------+---------------------------------+------------------+
| 72-1c /         | The formulated product  (23.6%  | LC~50~ eup  96  |
| 432975-01       | acid blue and 2.39% acid yellow | mg/L             |
|                 | dyes) was placed in the water   |                  |
| Acute coldwater | of aquaria. The study           | NOAEC eup =      |
| fish-           | concentration was not adjusted  |                  |
|                 | for the purity of the active    | 96 mg/L          |
| Rainbow         | ingredients (the two dyes). The |                  |
| trout ^1^       | results are milligrams of the   |                  |
|                 | end-use product. The only dose  |                  |
|                 | used was 96 mg/L.               |                  |
+-----------------+---------------------------------+------------------+
| 72-1a /         |                                 |                  |
| 432975-02       |                                 |                  |
|                 |                                 |                  |
| Acute warmwater |                                 |                  |
| fish- bluegill  |                                 |                  |
| sunfish ^1^     |                                 |                  |
+-----------------+---------------------------------+------------------+
| 72-2a /         | The formulated product  (23.6%  | LC~50~ eup  97  |
| 432975-03       | acid blue and 2.39% acid yellow | mg/L             |
|                 | dyes) was placed in the water   |                  |
| Freshwater      | of aquaria. The study           | NOAEC eup =      |
| invertebrate    | concentration was not adjusted  |                  |
| *Daphnia magna* | for the purity of the active    | 97 mg/L          |
|  ^1^            | ingredients (the two dyes). The |                  |
|                 | results are milligrams of the   |                  |
|                 | end-use product. The only dose  |                  |
|                 | used was 97 mg/L.               |                  |
+-----------------+---------------------------------+------------------+
| 72-1a           | Freshwater cladocerans          | EC~50~ =         |
|                 | (*Ceriodaphnia dubia*) were     | 5707 mg/L        |
| Warne &         | exposed to a dye for two days.  |                  |
| Schifko, 1999   | The EC~50~ put the chemical in  |                  |
|                 | the category "practically       |                  |
| FW Cladoceran   | nontoxic."                      |                  |
+-----------------+---------------------------------+------------------+
| 72-3a           | Exposure to marine habitats not | N/A              |
|                 | expected                        |                  |
| E               |                                 |                  |
| stuarine/Marine |                                 |                  |
| Acute Toxicity  |                                 |                  |
| with Sheepshead |                                 |                  |
| Minnow, Oyster, |                                 |                  |
| Mysids          |                                 |                  |
+-----------------+---------------------------------+------------------+
| 72-4a           | Exposure to marine habitats not | N/A              |
|                 | expected                        |                  |
| Freshwater Fish |                                 |                  |
| Early Life      |                                 |                  |
| Stage Fathead   |                                 |                  |
| minnow          |                                 |                  |
+-----------------+---------------------------------+------------------+
| 72-4b           | Chronic effects not expected    | N/A              |
|                 | based on mode of action and     |                  |
| Freshwater      | lack of effects in acute tests. |                  |
| Invertebrate    |                                 |                  |
| Life Cycle-     |                                 |                  |
| Water flea      |                                 |                  |
+-----------------+---------------------------------+------------------+
| 123-1a          | If the label is changed and the | Reserved         |
|                 | water is not used for           |                  |
| Tier II         | irrigation, exposure to         |                  |
| Terrestrial     | terrestrial plants would be     |                  |
| Plant Seedling  | unlikely.                       |                  |
| Emergence (GF   |                                 |                  |
| 871)            |                                 |                  |
+-----------------+---------------------------------+------------------+
| 123-1b          | If the label is changed and the | Reserved         |
|                 | water is not used for           |                  |
| Tier II         | irrigation, exposure to         |                  |
| Terrestrial     | terrestrial plants would be     |                  |
| Plant           | unlikely.                       |                  |
| Vegetative      |                                 |                  |
| Vigor (GF 871)  |                                 |                  |
+-----------------+---------------------------------+------------------+
| 123-2           | Aquatic plants are the targets. | N/A              |
|                 | Therefore, testing is not       |                  |
| Tier II Aquatic | required. Release from treated  |                  |
| Plant Studies   | ponds to other water bodies     |                  |
|                 | considered a very low           |                  |
|                 | probability.                    |                  |
+-----------------+---------------------------------+------------------+
| 141-1           | Mode of action suggests low     | N/A              |
|                 | likelihood of toxicity. The     |                  |
| Honey Bee Acute | method of application minimizes |                  |
| Contact         | exposure to terrestrial         |                  |
| Toxicity        | insects.                        |                  |
+-----------------+---------------------------------+------------------+
| Nonguideline    | Crayfish were exposed to 3      | NOAEL = 15 mg/L  |
|                 | levels of Aquashade for 5 days. |                  |
| Spencer, 1984   | The oxygen use was effected.    |                  |
|                 | The study was categorized as    |                  |
| Crayfish        | "slightly toxic."               |                  |
+-----------------+---------------------------------+------------------+
| Nonguideline    | Laboratory rats were fed 4      | NOAEL =          |
|                 | levels of yellow 5 for 900      |                  |
| Borzelleca and  | days. The study was categorized | 984 mg/kg/day    |
| Hallagan, 1988. | as "practically nontoxic."      |                  |
| Chronic         |                                 | LOAEL =  2641    |
| toxicity/       |                                 | mg/kg/day        |
| carcinogenicity |                                 |                  |
| ^3^             |                                 |                  |
+-----------------+---------------------------------+------------------+
| Nonguideline    | House mice (*Mus musculus*)     | 7354 mg/kg/day   |
|                 | were given food with 3 levels   |                  |
| Borzelleca, *et | of dye in their diet for 720    |                  |
| al.*, 1990      | days. The study was             |                  |
|                 | characterized as "practically   |                  |
| Chronic         | nontoxic."                      |                  |
| toxicity/       |                                 |                  |
| carcinogenicity |                                 |                  |
| in mice ^3^     |                                 |                  |
+-----------------+---------------------------------+------------------+
| ^1^ 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.      |                                 |                  |
|                 |                                 |                  |
| ^2^ The study   |                                 |                  |
| was done with   |                                 |                  |
| Admiral Lake    |                                 |                  |
| Colorant WSP.   |                                 |                  |
| The LD~50~ 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 LD~50~ |                                 |                  |
| a.i. cannot be  |                                 |                  |
| determined, but |                                 |                  |
| is probably 50% |                                 |                  |
| of the stated   |                                 |                  |
| \>5,000 mg/kg.  |                                 |                  |
|                 |                                 |                  |
| ^3^ The 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) except the acute mammal LD~50~ was done with Admiral WSP and
the reproductive mammalian 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 being 13% pure. To demonstrate the
degree of uncertainty, the toxicity values may be multiplied by 0.1 to
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 Type^1^**      **^Source^**   **^Extrapolated\ Endpoint^**

  ^Bobwhite\ quail^     ^Acute\ Avian\ Oral^   ^433367-01^    ^LD^~50~
                                                              ^&\ NOAEL\ \ 2250\ x\ 0.13\ \ 292.5\ mg/kg^

  ^Mallard\ duck^       ^Acute\ Avian\ Oral^   ^433367-02^    ^LD^~50~
                                                              ^&\ NOAEL\ \ 2250\ x\ 0.13\ \ 292.5\ mg/kg^

  ^Bobwhite\ quail^     ^Avian\ Dietary^       ^435034-03^    ^LC^~50~
                                                              ^&\ NOAEL\ \ 620\ x\ 0.13\ \ 80.6\ ppm^

  ^Mallard\ duck^       ^Avian\ Dietary^       ^435034-04^    ^LC^~50~
                                                              ^&\ NOAEL\ \ 620\ x\ 0.13\ \ 80.6\ ppm^

  ^Bluegill\ sunfish^   ^Acute\ Toxicity^      ^432975-02^    ^LC^~50~
                                                              ^&\ NOAEC\ \ 96\ x\ 0.13\ \ 12.48\ mg/L^

  ^Rainbow\ trout^      ^Acute\ Toxicity^      ^432975-01^    ^LC^~50~
                                                              ^&\ NOAEC\ \ 96\ x\ 0.13\ \ 12.48\ mg/L^

  *^Daphnia\ magna^*    ^Acute\ Toxicity^      ^432975-03^    ^LC^~50~
                                                              ^&\ NOAEC\ \ 97\ x\ 0.13\ \ 12.61\ mg/L^

  ^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 NOAEL  146
mg/kg and aquatic organism NOAEC 6.3 mg/L.

**Appendix E Status of Data Requirements**

1.  *Environmental Fate*

Subdivision N, Environmental Fate Data Requirements- Status of Data
Requirements

+-------------------------------+------------+--------------+--------+
| **Data Requirement**          | **Status   | **Status     | **Addi |
|                               | in 1993**  | after the    | tional |
|                               |            | Risk         | Data   |
|                               |            | Assessment   | Requ   |
|                               |            | for          | ired** |
|                               |            | Rere         |        |
|                               |            | gistration** |        |
+===============================+============+==============+========+
| 161-1 \[Abiotic\] Hydrolysis  | Reserved   | May be       | No     |
|                               |            | Waived       |        |
+-------------------------------+------------+--------------+--------+
| 161-2 \[Direct\] Photolysis   | Reserved   | May be       | No     |
| in Water                      |            | Waived       |        |
+-------------------------------+------------+--------------+--------+
| 161-3 Photolysis on Soil      | Not        | Not Required | No     |
|                               | Required   |              |        |
+-------------------------------+------------+--------------+--------+
| 162-1 Aerobic Soil Metabolism | Not        | Not Required | No     |
|                               | Required   |              |        |
+-------------------------------+------------+--------------+--------+
| 162-2 Anaerobic Aquatic       | Not        | Not Required | No     |
| Metabolism                    | Required   |              |        |
+-------------------------------+------------+--------------+--------+
| 162-3 Anaerobic Aquatic       | Reserved   | May be       | No     |
| Metabolism                    |            | Waived       |        |
+-------------------------------+------------+--------------+--------+
| 162-4 Aerobic Aquatic         | Reserved   | May be       | No     |
| Metabolism                    |            | Waived       |        |
+-------------------------------+------------+--------------+--------+
| 163-1 Mobility in             | Reserved   | May be       | No     |
| Soils/Sediments               |            | Waived       |        |
+-------------------------------+------------+--------------+--------+
| 164-1 Terrestrial Field       | Not        | Not Required | No     |
| Dissipation                   | Required   |              |        |
+-------------------------------+------------+--------------+--------+
| 164-2 Aquatic Field           | Reserved   | May be       | No     |
| Dissipation                   |            | Waived       |        |
+-------------------------------+------------+--------------+--------+
| 165-4 Bioaccumulation in Fish | Reserved   | May be       | No     |
|                               |            | Waived       |        |
|                               |            |              |        |
|                               |            | The Log Kow  |        |
|                               |            | does not     |        |
|                               |            | trigger this |        |
|                               |            | data         |        |
|                               |            | requirement  |        |
+-------------------------------+------------+--------------+--------+

+-------+------------------------+----------+--------+------+-------+
| **    |                        |          |        |      |       |
| Ecolo |                        |          |        |      |       |
| gical |                        |          |        |      |       |
| ef    |                        |          |        |      |       |
| fects |                        |          |        |      |       |
| data  |                        |          |        |      |       |
| re    |                        |          |        |      |       |
| quire |                        |          |        |      |       |
| ments |                        |          |        |      |       |
| for   |                        |          |        |      |       |
| pest  |                        |          |        |      |       |
| icide |                        |          |        |      |       |
| re    |                        |          |        |      |       |
| gistr |                        |          |        |      |       |
| ation |                        |          |        |      |       |
| of    |                        |          |        |      |       |
| A     |                        |          |        |      |       |
| quash |                        |          |        |      |       |
| ade** |                        |          |        |      |       |
|       |                        |          |        |      |       |
| PC    |                        |          |        |      |       |
| Code: |                        |          |        |      |       |
| 1     |                        |          |        |      |       |
| 10303 |                        |          |        |      |       |
| (a    |                        |          |        |      |       |
| mi    |                        |          |        |      |       |
| xture |                        |          |        |      |       |
| of    |                        |          |        |      |       |
| 1     |                        |          |        |      |       |
| 10301 |                        |          |        |      |       |
| and   |                        |          |        |      |       |
| 110   |                        |          |        |      |       |
| 302), |                        |          |        |      |       |
| only  |                        |          |        |      |       |
| TEPs  |                        |          |        |      |       |
| were  |                        |          |        |      |       |
| st    |                        |          |        |      |       |
| udied |                        |          |        |      |       |
+=======+========================+==========+========+======+=======+
| Guid  | DATA REQUIREMENTS      | FULFILLS | MRID   | DATE | S     |
| eline |                        | REQ\'S   |        |      | TATUS |
| N     | (Not Reserved)         |          |        |      |       |
| umber |                        | (Y/N)    |        |      |       |
+-------+------------------------+----------+--------+------+-------+
| 71    | Acute Avian Oral Quail | Y        | 433    | 9-   | Accep |
| -1(b) | TEP                    |          | 367-01 | 7-94 | table |
+-------+------------------------+----------+--------+------+-------+
|       | Acute Avian Oral Duck  | Y        | 433    | 9-   | Accep |
|       | TEP                    |          | 367-02 | 7-94 | table |
+-------+------------------------+----------+--------+------+-------+
| 71    | Avian Dietary/Quail    | Y        | 435    | 4-2  | Accep |
| -2(b) | TEP                    |          | 034-03 | 6-95 | table |
+-------+------------------------+----------+--------+------+-------+
| 71    | Avian Dietary/Duck TEP | Y        | 435    | 4-2  | Accep |
| -2(b) |                        |          | 034-04 | 6-95 | table |
+-------+------------------------+----------+--------+------+-------+
| 71-3  | Wild Mammal            | Y        | 452    | 12-  | Accep |
|       | Toxicity^a^            |          | 811-01 | 5-00 | table |
+-------+------------------------+----------+--------+------+-------+
| 71    | Avian                  | Waived   |        |      |       |
| -4(a) | Reproductive/Quail     |          |        |      |       |
+-------+------------------------+----------+--------+------+-------+
| 71    | Avian                  | Waived   |        |      |       |
| -4(b) | Reproductive/Duck      |          |        |      |       |
+-------+------------------------+----------+--------+------+-------+
| 72    | Acute Toxicity         | Y        | 432    | 4-2  | Accep |
| -1(b) | Bluegill TEP           |          | 975-02 | 5-95 | table |
+-------+------------------------+----------+--------+------+-------+
| 72    | Acute Toxicity Rainbow | Y        | 432    | 4-2  | Accep |
| -1(d) | Trout TEP              |          | 975-01 | 5-95 | table |
+-------+------------------------+----------+--------+------+-------+
| 72    | Acute Invertebrate     | Y        | 432    | 4-2  | Accep |
| -2(b) | Toxicity TEP           |          | 975-03 | 5-95 | table |
+-------+------------------------+----------+--------+------+-------+
| ^a^   |                        |          |        |      |       |
| This  |                        |          |        |      |       |
| study |                        |          |        |      |       |
| was   |                        |          |        |      |       |
| perf  |                        |          |        |      |       |
| ormed |                        |          |        |      |       |
| with  |                        |          |        |      |       |
| Ad    |                        |          |        |      |       |
| miral |                        |          |        |      |       |
| WSP   |                        |          |        |      |       |
+-------+------------------------+----------+--------+------+-------+

# No additional effects data are being required. 

# Appendix F Environmental Fate Bibliography

[Books]{.underline}

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.

[Journal Articles]{.underline}

Jank. M., Köser, H., Lücking, F., Martienssen, M., and Wittchen, S.
1998, "*Decolorization and Degradation of Erioglaucine (Acid Blue 9) Dye
in Wastewater,"* [Environmental Technology]{.underline}, v.19(7),
pp.741-747.

Weber, E.J. and Adams, R.L. 1995. *"Chemical and Sediment-Mediated
Reduction of the Azo* *Dye Disperse Blue 79,\"* [Environ. Sci.
Technol]{.underline}. v.29, pp. 1163-1170.

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 FeCl~3~ on
the photocatalytic degradation of dissolved azo dyes in aqueous TiO~2~
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.

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

Number range CHAPTER

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

Number range CHAPTER

**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 granular/bait 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) LC~50~ (fish and birds), (2) LD~50~ (birds
and mammals), (3) EC~50~ (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^1^/LC50 or LD50/sqft^2^ or LD50/day^3^ 0.5

  Acute Restricted Use EEC/LC50 or LD50/sqft or LD50/day (or LD50 0.2
                       \< 50 mg/kg)                               

  Acute Endangered     EEC/LC50 or LD50/sqft or LD50/day          0.1
  Species                                                         

  Chronic Risk         EEC/NOAEC                                  1

  Wild Mammals                                                    

  Acute High Risk      EEC/LC50 or LD50/sqft or LD50/day          0.5

  Acute Restricted Use EEC/LC50 or LD50/sqft or LD50/day (or LD50 0.2
                       \< 50 mg/kg)                               

  Acute Endangered     EEC/LC50 or LD50/sqft or LD50/day          0.1
  Species                                                         

  Chronic Risk         EEC/NOAEC                                  1
  -------------------------------------------------------------------------

^1^ abbreviation for Estimated Environmental Concentration (ppm) on
avian/mammalian food items

^2^ mg/ft^2^

^3^ 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^1^/LC50 or EC50            0.5

  Acute Restricted Use            EEC/LC50 or EC50               0.1

  Acute Endangered Species        EEC/LC50 or EC50               0.05

  Chronic Risk                    EEC/MATC 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^1^/EC25                    1

  Acute Endangered Species        EEC/EC05 or NOAEC              1

  Aquatic Plants                                                 

  Acute High Risk                 EEC^2^/EC50                    1

  Acute Endangered Species        EEC/EC05 or NOAEC              1
  ------------------------------- ------------------------------ ---------

^1^ EEC = lbs ai/A

^2^ EEC = (ppb/ppm) in water

Number range CHAPTER

Number range CHAPTER**Appendix L. Papers that were accepted for ECOTOX
but that were not used in the risk assessment science chapter. These
papers passed the screen for consideration in ecological risk
assessment, but were ultimately not used because they did not add to or
change the risk presumption.**

**Acceptable for ECOTOX and OPP**

Borzelleca, J. F., Depukat, K., and Hallagan, J. B. 1990. Lifetime
Toxicity/Carcinogenicity Studies of FD-and-C Blue No. 1 (Brilliant Blue
FCF) in Rats and Mice. *Food Chem.Toxicol.* 28: 221-234. EcoReference
No.: 76018\
Chemical of Concern: AQS; Habitat: T; Effect Codes:
GRO,POCM,MOR,BEH,CEL.

Borzelleca, J. F. and Hallagan, J. B. 1988. A Chronic
Toxicity/Carcinogenicity Study of FD & C Yellow No. 5 (Tartrazine) in
Mice. *Food Chem.Toxicol.* 26: 189-194.EcoReference No.: 76019\
Chemical of Concern: AQS; Habitat: T; Effect Codes: MOR,GRO,BEH,CEL.

Collins, T. F. X., Black, T. N., Brown, L. H., and Bulhack, P. 1990.
Study of the Teratogenic Potential of FD and C Yellow No. 5 when Given
by Gavage to Rats. *Food Chem.Toxicol.* 28: 821-827. EcoReference No.:
76021\
Chemical of Concern: AQS; Habitat: T; Effect Codes: GRO,BEH,PHY,REP,MOR.

Collins, T. F. X., Black, T. N., O\'Donnell, M. W. Jr., and Bulhack, P.
1992. Study of the Teratogenic Potential of FD & C Yellow No. 5 when
Given in Drinking-Water. *Food*

*Chem.Toxicol.* 30: 263-268.EcoReference No.: 76022\
Chemical of Concern: AQS; Habitat: T; Effect Codes: GRO,BEH,PHY,REP,MOR.

Ershoff, B. H. 1977. Effects of Diet on Growth and Survival of Rats Fed
Toxic Levels of Tartrazine (FD & C Yellow No. 5) and Sunset Yellow FCF
(FD & C Yellow No. 6. *J.Nutr.* 107: 822-828. EcoReference No.: 76051\
Chemical of Concern: AQS; Habitat: T; Effect Codes: GRO,MOR.

Giri, A. K., Das, S. K., Talukder, G., and Sharma, A. 1990. Sister
Chromatid Exchange and Chromosome Aberrations Induced by Curcumin and
Tartrazine on Mammalian Cells In Vivo. *Cytobios* 62: 111-117.
EcoReference No.: 76048\
Chemical of Concern: AQS; Habitat: T; Effect Codes: CEL.

Maekawa, A., Matsuoka, C., Onodera, H., Tanigawa, H., Furuta, K., Kanno,
J., Jang, J. J., Hayashi, Y., and Ogiu, T. 1987. Lack of Carcinogenicity
of Tartrazine (FD and C Yellow No. 5) in the F344 Rat. *Food
Chem.Toxicol.* 25: 891-896. EcoReference No.: 76017\
Chemical of Concern: AQS; Habitat: T; Effect Codes: CEL,GRO,MOR.

Sasaki, Y. F., Kawaguchi, S., Kamaya, A., Ohshita, M., Kabasawa, K.,
Iwama, K., Taniguchi, K., and Tsuda, S. 2002. The Comet Assay with 8
Mouse Organs: Results with 39 Currently Used Food Additives.
*Mutat.Res.* 519: 103-119. EcoReference No.: 75840\
Chemical of Concern: AQS,TBA,TRZ,BZO; Habitat: T; Effect Codes: MOR,CEL.

Sobotka, T. J., Brodie, R. E., and Spaid, S. L. 1977. Tartrazine and the
Developing Nervous System of Rats. *J.Toxicol.Environ.Health* 2:
1211-1220. EcoReference No.: 76050\
Chemical of Concern: AQS; Habitat: T; Effect Codes: PHY,BEH,GRO,BCM.

Stefanidou, M., Alevisopoulos, G., Chatziioannou, A., and Koutselinis,
A. 2003. Assessing Food Additive Toxicity Using a Cell Model.
*Vet.Hum.Toxicol.* 45: 103-105. EcoReference No.: 76016\
Chemical of Concern: AQS,NaNO3; Habitat: A; Effect Codes: CEL.

Tripathy, N. K., Patnaik, K. K., and Nabi, M. J. 1989. Genotoxicity of
Tartrazine Studied in Two Somatic Assays of Drosophila melanogaster.
*Mutat.Res.* 224: 479-483. EcoReference No.: 76049\
Chemical of Concern: AQS; Habitat: T; Effect Codes: CEL.

Biggs, D. F., Peterson, M. A., and Aaron, T. H. 1981. Tartrazine Induced
Airway Constriction in Anesthetized, Paralyzed Guinea-Pigs.
*Proc.West.Pharmacol.Soc.* 24: 363-365. EcoReference No.: 75992\
Chemical of Concern: AQS; Habitat: T; Effect Codes: PHY.

D\'Souza, S. J. A. and Biggs, D. F. 1987. Aspirin, Indomethacin, and
Tartrazine Increase Carotid-Sinus-Nerve Activity and Arterial Blood
Pressure in Guinea Pigs. *Pharmacology* 34: 96-103. EcoReference No.:
76170\
Chemical of Concern: AQS; Habitat: T; Effect Codes: PHY.

Davis, K. J., Fitzhugh, O. G., and Nelson, A. A. 1964. Chronic Rat and
Dog Toxicity Studies on Tartrazine. *Toxicol.Appl.Pharmacol.* 6:
621-626. EcoReference No.: 76249\
Chemical of Concern: AQS; Habitat: T; Effect Codes: MOR,CEL,GRO,PHY.

Kobayashi, N., Taniguchi, N., Sako, F., and Takakuwa, E. 1977. A
Screening Method for the Toxicity of Food Dyes Using Artemia salina
Larvae. *J.Toxicol.Sci.* 2: 383-390. EcoReference No.: 7542\
Chemical of Concern: AQS; Habitat: A; Effect Codes: MOR.

Manker, D. C. and Martin, D. F. 1984. Investigation of Two Possible
Modes of Action of the Inert Dye Aquashade on Hydrilla.
*J.Environ.Sci.Health Part A* 19: 725-733. EcoReference No.: 11634\
Chemical of Concern: AQS; Habitat: A; Effect Codes: GRO.

Nicklin, S. and Miller, K. 1985. Induction of a Transient Reaginic
Antibody to Tartrazine in an Animal Model. *Int.Arch.Allergy
Appl.Immunol.* 76: 185-187. EcoReference No.: 76218\
Chemical of Concern: AQS; Habitat: T; Effect Codes: PHY.

Office of Pesticide Programs 2000. Pesticide Ecotoxicity Database
(Formerly: Environmental Effects Database (EEDB). *Environmental Fate
and Effects Division, U.S.EPA, Washington, D.C.* EcoReference No.: 344\
Chemical of Concern:
24DXY,ACL,ACP,ACR,AQS,ATZ,AZ,BDF,BMC,BML,BMN,BS,BT,Captan,CBF,CBL,CFE,CFE,CLNB,CMPH,CPC,CPY,CTN,CTZ,Cu,CuO,CuS,CYD,CYF,CYP,CYT,DBN,DCNA,DFT,DFZ,DM,DMB,DMM,DMP,DMT,DOD,DPC,DPDP,DS,DSP,DU,DZ,DZM,EFL,EFS,EFV,EP,FHX,FMP,FO,Folpet,FPP,FVL,GYP,HCCH,HXZ,IPD,IZP,LNR,MAL,MB,MBZ,MDT,MFX,MFZ,MGK,MLN,MLT,MOM,MP,MTC,MTL,MTM,NAA,Naled,NFZ,NPP,NTP,OXF,OXT,OYZ,PDM,PEB,PHMD,PMR,PMT,PNB,PPB,PPG,PPMH,PQT,PRB,PRT,PSM,PYN,PYZ,RTN,SMM,SMT,SS,SXD,SZ,TBC,TDC,TDZ,TET,TFN,TFR,TMT,TPR,TRB,WFN,ZnP;
Habitat: AT; Effect Codes: MOR,POP,PHY,GRO,REP.

Peterson, M. A., Biggs, D. F., and Aaron, T. H. 1980. Comparison of the
Effects of Aspirin, Indomethacin and Tartrazine on Dynamic Pulmonary
Compliance and Flow Resistance in the Guinea Pig.
*Proc.West.Pharmacol.Soc.* 23: 121-124. EcoReference No.: 75980\
Chemical of Concern: TRZ,AQS; Habitat: T; Effect Codes: CEL,PHY.

Procter & Gamble Co. 1992. Initial Submission: Repeated Dermal
Applications in Mice of FD&C Blue 1, FD&C Red 3, FD&C Yellow 5, D&C Red
19 with Cover Letter Dated 08/26/92. *EPA/OTS Doc.#88-920008654*.
EcoReference No.: 76262\
Chemical of Concern: AQS; Habitat: T; Effect Codes: PHY,MOR.

Safford, R. J. and Goodwin, B. F. J. 1985. Immunological Studies on
Tartrazine and Its Metabolites 1. Animal Studies. *Int.Arch.Allergy
Appl.Immunol.* 77: 331-336. EcoReference No.: 76217\
Chemical of Concern: AQS; Habitat: T; Effect Codes: PHY.

Sako, F., Taniguchi, N., Kobayashi, N., and Takakuwa, E. 1977. Effects
of Food Dyes on Paramecium caudatum: Toxicity and Inhibitory Effects on
Leucine Aminopeptidase and Acid Phosphatase Activity.
*Toxicol.Appl.Pharmacol.* 39: 111-117 (Author Communication Used.

EcoReference No.: 8261\
Chemical of Concern: AQS; Habitat: A; Effect Codes: MOR.

Schafer, E. W. and Bowles, W. A. 1985. Acute Oral Toxicity and
Repellency of 933 Chemicals to House and Deer Mice.
*Arch.Environ.Contam.Toxicol.* 14: 111-129.

EcoReference No.: 35426\
Chemical of Concern: ADC,CST,MOM,CPC,ZnP,DOD,MLN,Cu,AQS,CuCO; Habitat :
T; Effect Codes: MOR.

Upadhyay, R. R. and Upadhyay, L. 1994. Cirrhosis and Dysplasia Caused by
Sun Set Yellow and Brilliant Blue in the Liver of Heteropneustes
fossilis. *J.Ecotoxicol.Environ.Monit.* 4: 275-277. EcoReference No.:
20309\
Chemical of Concern: AQS; Habitat: A; Effect Codes: HIS.

Wan, M. T., Watts, R. G., and Moul, D. J. 1991. Acute Toxicity to
Juvenile Pacific Northwest Salmonids of Basacid Blue NB755 and Its
Mixture with Formulated Products of 2,4-D, Glyphosate, and Triclopyr.
*Bull.Environ.Contam.Toxicol.* 47: 471-478 (OECDG Data File.
EcoReference No.: 5132\
Chemical of Concern: 24DXY,GYP,TPR,AQS; Habitat: A; Effect Codes: MOR.

[^1]: The specific names used for "Acid Blue 9\"under the FDCA are "FD&C
    Blue No.1\" for the trisodium 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.

[^2]: 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 is a combination of two
    dyes, each with and assigned PC Number,

[^3]: EPI (Estimation Programs Interface) Suite^TM^ (formerly known as
    EPIWIN)

[^4]: 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.

[^5]: "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.

[^6]: "Reassessment of the Exemption from the Requirement of a Tolerance
    for the FDA-Certified 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 21, 2004.

[^7]: http://www.colour‑index.org/

[^8]: Light is energy. The color observed in dyes (or in gems) is caused
    by electronic transitions between energy levels of a molecule. The
    color-giving absorption band is known as a chromophore ("color
    bearer")

[^9]: Although Acid Blue 9 and Acid Yellow 23 belong to different
    chemical families, both dyes contain sulfonate groups.

[^10]: 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, 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.

[^12]: In artist's color language, a dye or pigment that fades is said
    to be "fugitive" and those that do not fade are called "permanent
    colors".

[^13]:

[^14]: Memorandum: "Review of Phase 4 List D Package for Aquashade"
    (EFGWB \# 93-0119, 93-0120; Chemical \# 110301 and 110302; Case \#
    819437 and 819438; DP Barcode D184289 and D184278). Dated February
    4, 1993.

[^15]: "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.

[^16]: Natural photosensitizers in environmental waters include
    dissolved organic matter (DOM), singlet oxygen (^1^O~2~), and
    hydroxy 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.

[^17]: See, for example, "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.