Page 1 of 21 OFFICE OF
CHEMICAL SAFETY AND
POLLUTION PREVENTION UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON D.C., 20460

PC C od e s : 110301 &

110302

DP B arcode : D425778

D ate : August 27 , 201 5
MEMORANDUM

SUBJECT: Problem Formulation and Preliminary R isk Assessment for Support of the Registration Review for Aqua tic Dyes Containing Acid Blue 9 ( PC Code

110301 ) and Acid Yellow 23 ( PC Code 110302 )

FROM: Nicholas Mastrota, Ph.D., Wildlife Biologist James K. Wolf, Ph.D., Environmental Scientist

Environmental Risk Branch 3 Environmental Fate and Effects Division ( 7507P)

REVIEWED Rosanna Louie

- Juzwiak, Risk Assessment Process Leader

BY: Melissa Panger, Ph.D., Senior Biologist James Hetrick, Ph.D., Senior Advisor

Environmental Risk Branch 3 Environmental Fate and Effects Division ( 7507P)

THROUGH: Dana Spatz, Branch Chief

Environmental Risk Branch 3 Environmental Fate and Effects Division ( 7507P)

TO: Neil Anderson , Bran ch Chief

Christina Motilall , Chemical Review Manager Risk Management and Implementation Branch 1

Pesticide Re

- Evaluation Division (7508P)

The Environmental Fate and Effects Division (EFED) ha s completed the preliminary ecological

risk and drinking water assessments in support of the registration review of the aquatic dyes containing a cid b lue 9 ( PC Code 110301 ) and acid yellow 23 ( PC Code 110302)

. Alternative

names for a cid b lue 9 and acid yellow 23 are erioglaucine and tartrazine, respectively. These dye mixtures are marketed under the trade names Aquashade ® (EPA Reg. # 33068

- 1, 33068

- 2, 33068

- 3) and Admiral ® (EPA Reg # 67064

- 1, 67064

- 2). Since uses of these dyes have not changed, and no new environmental fate or ecotoxicity testing has been submitted, little has changed since these dyes were previously assessed for reregistration in 2005. Therefore, this abbreviated risk assessment provides updates to exposure and risk conclusions not previously assessed ( i.e.

, chronic risk to mamma ls) . This document serves as both the problem formulation and thePage 2 of 21 preliminary risk assessment in support of the registration review of aquatic dyes containing Acid

Blue 9 and Acid Yellow 23.Page 3 of 21 

### 1. Executive summary

The Environmental Fate and Effects Division ( EFED) h as completed the preliminary ecological

risk and drinking water assessments in support of the registration review of the aquatic dye s containing Acid Blue 9 ( PC Code 110301 ) and Acid Yellow 23 ( PC Code 110302)

. No new environmental fate or ecotoxicity testing has been submitted since the ecological risk assessment

was conducted in 2005 for reregistration of these dyes. Also, no new uses or products have been added since that time. The restriction of these dyes to water bodies “under complete control of the user and have little or no outflow” and the prohibition of use in water that may be used for human consumption greatly limits exposure to drinking water resources or aquatic habitats other than

those treated dir ectly. Exposure to drinking water resources from registered use of these dyes is

also believed to be negligible. The available ecotoxicity data, together with information on risks to aquatic organisms available from the open literature, are sufficient to a ssess ecological risks. Furthermore, all available toxicity information indicates that these dyes have very low toxicity and pose little risk to animals and plants at environmentally relevant concentrations.



### 2. Previous Assessments

EFED ’s most recent ecolog ical risk assessment for dyes containing Acid Blue 9 and Acid Yellow

23 was completed for the previous reregistration process and was summarized in a Reregistration Eligibility D ecision (RED 1 ) from September 26, 2005 (D302183). Since that memorandum was

co mpleted, the only new information that EFED has obtained that is relevant to the ecological risk of these dyes ha s come from open literature papers. Several papers have been published on the toxicity of these dyes to aquatic organisms (or lack thereof), an d their effects to fish, plankton, and aquatic plants when used in aquaculture system s . Therefore, much of this assessment consists of a review and summary of findings from these published papers, and a discussion of how this

new information effects the ri sk conclusions that EFED ha d previously reported f or use of these dyes

.



### 3. Nature of Chemical Stressor

Th e chemical stressor s are mixtures of acid blue 9 (also known as erioglaucine) and acid yellow 23 (also known as tartrazine) that are used to color small water bod ies blue

- green to attenuate light penetration, and thereby reduce growth of nuisance aquatic plants and algae

. While the proportions of the two dyes vary somewhat among the four registered products, a cid blue 9 is

alw ays by far the larger component, comprising 92% to 94% of the total dye by weight. Ratios of acid blue 9 to acid yellow 23 range from 12.1:1 to 15.3:1 ( Table 2 ). These ratios of these products are similar enough that the environmental exposure and toxicity of the different mixtures of the dyes in the four registered products will be assumed to be equivalent.

1 USEPA. 2005. Reregistration Eligibility D ecision (RED) Ecological Risk Assessment of End

- Use Products

Containing the Dyes Acid Blue 9 and Acid Yellow 23. http://www.epa.gov/pesticides/r eregistration/REDs/aquashade_red.pdfPage 4 of 21 Aquatic dyes containing acid blue 9 and acid yellow 23 are used to color water in small bodies of water to inhibit growth of algae and submerged aquat ic weeds on the bottom. These dyes turn the
water an aqua

- blue color, and in doing so filter out light of wavelengths that are used by plants for photosynthesis. Specificall y, the two dyes in aquashade absorb light primarily in the red

- orange and blue

- viol et parts of the spectrum, which is part of the spectrum used by aquatic plants and algae for photosynthesis (Lonza Corporation 2015, Spencer 1984). Light needed for photosynthesis is filtered out of the water, thereby reducing the amount of light reaching algae and submerged aquatic plants, especially in deeper water. These dyes are also used as “inert ingredients” in other pesticide formulations.

These two dyes are also regulated by the Food and Drug Administration (FDA) under the Food, Drugs, and Cosmetics Act (FDCA)

, and they have been approved for use as colorants for food

, drugs, and cosmetics, many of which are listed in the Household Product Database (USDHHS,

2014 2 )

. Acid yellow 23 is a synthetic lemon yellow dye primarily used as a food coloring . A number of personal care, cosmetics pr oducts, various types of medications and pharmaceutical products may include tartrazine. Other products, such as household cleaning products, paper plates, pet foods

, crayons , inks for writing instruments, stamp dyes, face paints and envelope glues, may also contain tartrazine a cid blue 9
, nonfood grade (acid blue 9) toilet bowl cleaner, laundry detergents and ( i.e. , the food grade Brilliant Blue FCF (Blue 1)), is often found in ice

cream
, canned processed peas , packet soups, bottled food colorings , icings, ice pops, b lue raspberry flavored p roducts, dairy products, sweets

, and drinks. It is also used in soaps
, shampoos

, mouthwash and other hygiene and cosmetics applications. In the area of soil

- water research, Brilliant Blue is applied in tracing studies to visualize infiltration and water distribution in the soil (Flury and Flühler, 1994). Tartrazine is a commonly used color all over the world, mainly for yellow, but can also be used with Brilliant Blue FCF or Acid blue 9 to produce various green shades. Only the pesticidal uses ( i.e ., those associated with aquatic weed control) will be

assessed here.

Ch emical names , struc tures, and other identifiers of acid blue 9 and acid yellow 23 are given in

Table 1

.

Table 1 Chemical Nomenclature and Structure of the Aquatic Dyes Acid Yellow 23 and Acid Blue 9.

Acid Yellow 23 Acid Blue 9

Common Name as Appearing in the

labels of the Admiral Products Tartrazine Erioglaucine (diammonium salt)

Empirical Formula C 16 H 9 N 4 Na 3 O 9 S 2 C 37 H 34 N 2 Na 2 O 9 S 3 (disodium salt)

C 37 H 34 N 2 (NH 4 ) 2 O 9 S 3 (diammonium salt)
Chemical Name(s) 4,5

- Dihydro

- 5

- oxo

- 1

- (4

- sulfophenyl)

- 4

- ((4

- sulfophenyl)azo)

- 1H

- pyrazole

- 3

- carboxylic acid trisodium salt

3

- carboxy

- 5

- hydroxy

- 1

- p

- sulfophenyl

- 4

- p

sulfophenylazopyrazole trisodium salt N

- Ethyl

- N

- (4

- [(4

- {ethyl[(3

- sulfophenyl)methyl]amino}phenyl)(2

- sulfophenyl)methylene]

- 2,5

- cyclohexadien

- l

- ylidene)

- 3

- sulfobenzenemethanaminium hydroxide inner salt, trisodium salt

N

- Ethyl

- N

- (4

- [(4

- {ethyl[(3

- sulfophe nyl)methyl]amino}phenyl)(2

-

2 US Department of Health and Human Service. Household Products Database. http://householdproducts.nlm.nih.gov/index.htmPage 5 of 21 Acid Yellow 23 Acid Blue 9

5

- hydroxy

- 1

- (p

- sulfophenyl)

- 4

- ((p

sulfophenyl) azo) pyrazole

- 3

- carboxy lic

acid trisodium salt sulfophenyl)methylene]

- 2,5

- cyclohexadien

- l

- ylidene)

- 3

- sulfobenzenemethanaminium hydroxide inner salt, triammonium

salt

Molecular Structure 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 t hat is responsible for the color of the dye.

Synonyms

FD & C Yellow No. 5:

21CFR74.705 (Foods) 21CFR74.1705 (Drugs) 21CFR74.2705 (Cosmetics) These FD&C dyes are of high purity and require batch certification analysis when used in food, drugs, or cosmetics.

CI Food Yellow 4

CI Number 15985

CAS Registry No. 1934

- 21

- 0 Sodium Salt : Brilliant Blue

CI Food Blue 2 Erioglaucine trisodium salt

CI Food Blue 2.

CI Number 42090 CAS Registry No. 2650

- 18

- 2

Triammonium salt Erioglaucine diammonium salt Alphazurine

CI Blue 8

CI 42090 CAS Reg. No. 3844

- 54

- 9

FD&C Blue 1 21CFR74.101 (Foods) 21CFR74.1101 (Drugs) 21CFR74.2101 (Cosmetics)

D&C Blue 4 21CFR74.1104 (Drugs) 21CFR.74.2104 (Cosmetics) Chemical Family Azo (monoazo) dye; Contains one azo

group,

- N=N

- ) Aminotriphenylmethane dye



### 4. Use Characterization

Aquatic dyes containing acid blue 9 and acid yellow 23 are used primarily in small water bodies like ornamental ponds and small lakes

, fountains and other landscaping water features, swimming holes, aquaculture ponds, and animal watering holes

. L abels of a ll end

- use products containing these dyes restrict use to water bodies that are “under complete control of the user and have little

or no outflow.” Use is permitted in hu man

- made water bodies, although adherence to the restrictions mentioned above would gr eatly restrict use in natural waters.Page 6 of 21 Table 2 summarizes the use information for the four registered products containing acid blue 9 and acid yellow 23

. All products are ready

- to

- use liquids except for Admiral WSP, which is a

water

- dispersible pellet. Products are applied directly into the water body from the shore or from a boat. Labels provide instructions for users to calculate the volume of the water body in units of

acre

- feet (acres of surface multiplied by average depth in feet), and provide instr uctions for how much product to apply per acre

- feet for various target concentrations of total dye. The maximum target concentration of total dye on all labels is 2.0 parts per million ( ppm )

.

Table 2 . Uses Site and Application Information for Aquatic Dye s Containing Acid Blue 9 and Acid Yellow 23.

Product Name (EPA Reg. No.) % AI Ratio Acid

Blue 9 : Acid

Yellow 23 Maximum Target

Concentration of Total Dye (ppm) Maximum Rate (lb/a

- ft*)

Acid Blue 9 Acid Yellow 23 Acid Blue

9 Acid

Yellow 23

Aquashade (33068

- 1) 12.60 1.04 12.1 : 1 2.0 0.6930 0.0572

Aquashade Plus (33068

- 3 ) 30.45 2.45 12.4 : 1 2.0 0.7316 0.0589

Admiral WSP (67064

- 1) 49.72 3.27 15.2 : 1 2.0 0.6837 0.0450

Admiral Liquid (67064

- 2) 15.31 1.00 15.3 : 1 2.0 0.6897 0.0451

* lb/a

- ft means pound s applied per the product of surface area in acres and mean water depth in feet.

The labels do not restrict reapplication of the product. Labels of Aquashade® products state:

“ Apply in season maintenance dosages, as needed, to restore loss of color due to dilution or dye degradation. ” Labels of Admiral® products state: “Reapply as needed during the growing

season.”



### 5. Conclusions from Previous Risk Assessments

In 2005, in support of reregistration, the Environmental Fate and Effects Division (EFED) completed a n ecological risk assessment for use of aquatic dyes containing acid blue 9 and acid yellow 23 to control aquatic weeds and algae (D302183 , 09/26/05 )

. In th at assessment, EFED

noted that b oth of these dyes are expected to be persistent in the environment, as dyes are generally not readily biodegradable under aerobic conditions and do not degrade via abiotic hydrolysis. Degradation is believed to occur by slow direct photolysis, especially in shallow water exposed to sunlight. The vapor pressure and the Henry’s Law Constant of dyes are very low, and thus little would volatilize from soil or water. Dyes such as acid blue 9 and acid yellow 23 are very hydrophilic (Log Kow << 1) and therefore are not likely to bioaccumulate in fish.

An aqua tic risk assessment was conducted based on aquatic animals being exposed to the maximum target concentrations of total dye specified on the label, 2 ppm. This was compared to results of acute aquatic toxicity studies conducted with Aquashade ® . Since expos ure at all test levels in the acute aquatic studies did not cause any mortality, conservative RQs were calculated as the EEC (2 ppm) divided by the observed NOAEC from the study. RQs for bluegill sunfish, rainbow trout, and Daphnia magna were all less than 0.01, indicating minimal acute risk to aquatic animals . For aquatic plants, risk to algae and submerged macrophytes in the treated pond is assumed since they are the target species of the products. However, since the labels limit use to

water bodies with little or no outlet, dye concentrations in the off

- site receiving bodies were expected to be very low and expected to have negligible effects on non

- target aquatic plants.Page 7 of 21 A terrestrial risk assessment was conducted based on predicted exposure to mammals a nd birds drinking from water treated with Aquashade ® at the maximum target concentration of 2 ppm. Water consumption was estimated based on allometric equations that predict daily water consumption for birds and for mammals based on bodyweight. For all wei ght classes of birds and mammals, acute RQs were less than 0.01, indicating minimal risk to terrestrial animals.

Calculations were also conducted to estimate exposure to birds and mammals if treated water were to be used for irrigation. Assuming that wate r containing 2 ppm were applied at a rate equivalent to 2 inches of irrigation, the rate of application of the dyes was determined to be approximately 1 lb a.i./A. A risk assessment for foliar application indicated that residues on plants

from this rate of applications would remain well below toxic levels.

Terrestrial plants were predicted to have negligible expos ure and risk from th e aquatic uses of these dyes.



### 6. Environmental Fate and Transport

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, birds or mammals, the requi rement for the submission of

environmental fate studies w as w aived

. 3 This assessment is primarily based upon the fate

information available from the last assessment. Open literature was reviewed for ad ditional or updated information and n o additional envir onmental fate data has been submitted since the last

assessment. The environmental fate data in this assessment are qualitative, based mostly on data from the open literature and on structurally

- related dyes which w ere reviewed in the previous assessment ( RED, USEPA, 2005). Although structure

- activity relationship (SAR) estimates using

EPI Suite/EPIWIN 4 were performed, these programs do not adequately estimate physical and chemical properties of salts, particularly those of a large anion such as in acid blu e 9 and acid yellow 23. A more detailed discussion concerning the chemistry and environmental fate of acid blue 9 and acid yellow 23 is located in the 2005 Reregistration Eligibility D ecision ( RED, USEPA, 2005). Table 3 summarizes the available information on the p roperties of a cid y ellow 23

and a cid b lue 9

.

Sources for data reviewed included USEPA High Production Volume Information System (HPVIS) Robust Summaries and Test Plan; C.I. Acid Yellow ( HPVIS, USEPA

, 2015 5 ) and the US National Library of Medicine, NIH, TOXNET (Acid blue dye 9 6 and A cid yellow dye 7 ) , and SRC (SRC, Inc, 2015)

. 8

The dissipation of acid blue 9 and acid yellow 23, the two active ingredients in Aquashade ® and

Admiral ® , will be due to a combination of dil ution, p hotodegradation, biodegradation, or seepage from the treated pond. Hydrolysis is not expected to be an important environmental fate process

3 USEPA. 2005. Ecological Risk As sessment of End

- Use Products Containing the Dyes Acid Blue 9 and Acid Yellow 23. (DP

Barcode D302183; 09/26/05).

4 EPI (Estimation Programs Interface) Suite TM (formerly known as EPIWIN).

5 http://www.epa.gov/hpv/pubs/summaries/ciacdylo/c15133tp.pdf

6 Acid Blue 9 http://toxnet.nlm .nih.gov/cgi

- bin/sis/search/a?dbs+hsdb:@term+@DOCNO+5890

7 Acid Yellow 23 http://toxnet.nlm.nih.gov/cgi

- bin/sis/search/a?dbs+hsdb:@term+@DOCNO+7216

8 SRC 2015. http://esc.syrres.com/fatepointer/search.aspPage 8 of 21 since these compounds lack functional groups that hydrolyze under environmental conditions.

Volatilization from moist soil surfaces is not expected to be an important fate process because the compound exists as an anion and anions do not volatilize. Volatilization from water surfaces is also not expected to be an important fate process based upon the ionic char acter of these compounds. The vapor pressures were also extremely low.

Photochemical degradation of Brilliant Blue FCF in aqueous solution can occur in surface waters,

such as lakes or rivers. For most dyes

, such degradation is likely to progress very slowly because synthetic dyes are designed to be very stable to light (Porter, 1973; Pagga and Brown, 1986; Zollinger, 1991; as cited by Flury and Flühler, 1994).

A “ dissipation half

- life ” of aquashade is repor ted to be four weeks and coloration is gradually lost by dilution, photodegradation and some biodegradation over time (Applied Biochemists Inc.,

1992 ; after Aquatic Dyes 9 ). However, as conditions among various bodies of water differ, there is a range of va riation in this estimate. In one study in which Aquashade was applied to earthen catfish ponds with high seepage rates, it was estimated that about one

- half of the dye applied initially was lost by seepage every 2 months ( Boyd and Noor, 1982). 10 They found that in a study that decline in Aquashade concentration in a catfish pond was primarily due to seepage suggesting that acid blue 9 and acid yellow 23 are quite persistent in an aquatic environment. In another study, the half

- life of Aquashade in a test pon d with no outlet was found to be about two months whereas in other test ponds, factors such as water outflow and evaporation affected the dye concentrations (White et al. , 1975 14 )

.

Both acid blue 9 and acid yellow 23 are expected to exist almost entirely in anion form in the

environment ; and anions generally do not adsorb more strongly to soils containing organic carbon and clay than their neutral counterparts and are therefore expected to be mobile in the environment. The mobility is support ed by the use of Brilliant blue FCF as a dye tracer for solute transport studies (Flury and Flühler, 1994).

Table 3

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

.

Information Acid Yellow 23 Acid Blue 9 Molecular Weight 534.37 (Marmion, 1991) 792.84 (disodium salt) 782.96 (diammonium salt) (Marmion, 1991)
Vapor pressure, at 25°C, in mmHg 7.43 x 10

- 22 there is some uncertainty in these estimated values, however they do not significantly impact the assessment as the vapor pressure are very low
EPI Suite, 2004; USEPA, 2004. HPVIS 11

25.0 (Reported in 43503402) 2.97 x 10

- 42 there is some uncertainty in these estimated values, however they do not significantly impact the assessment as the vapor pressur e are very low
EPI Suite, 2004

28.6 (Reported in 43503401)

9 http://www.mass.gov/eea/docs/agr/pesticides/aquatic/aquatic

- dyes.pdf

11 http://www.epa.gov/hpv/pubs/summaries/ciacdylo/c15133tp.pdfPage 9 of 21 Information Acid Yellow 23 Acid Blue 9 Solubility in Water, 25°C (mg/L) 1 x 10 6 ; EPI Suite, 2004 “Completely soluble” (Reported in 43503402) 200,000 mg/mL ( or mg/L ) USEPA,

2004. HPVIS 16 1.45 mg/L; EPI Suite, 2004 “Completely soluble” (Reported in 43503401)

Log n

- Octanol/Water

Partition Coefficient - 10.7 EPI Suite, 2004 and SRC

- 10.17 SCCNFP/0786/04

- 10.17 HPV IS 16 c1533rr2.pdf 201

- 16674B - 1.50 EPI Suite, 2004 and SRC

- 0.32 SCCNFP/0787/04

Log BCF

- 0.54 to 0.48 (MITI, 1992 ; after Ollgaard et al. 1998 12

. ) -

Terrestrial Fate Not biodegradable in activated sludge HPV c1533rr2.pdf 201

- 16674B -



### 7. Ecotoxicity

a. Ecotoxicity Data

Available ecotoxicity data for aquatic dyes containing acid blue 9 and acid yellow 23 are

summarized in Table 4 . The data are limited to tests of acute toxicity to terrestrial and aquatic

animals. All of the studies were classified as Acceptable . While the studies were conducted with one formulated product, the percentages of the two dyes in the other formulated products are similar enough to this one that the conclusions are assumed to be applicable to all registered products.

Acute oral and subacute dietary studies with birds found no mortality or sublethal effects at the limit dose or concentration. One acute oral study with the rat also found no mortality at the limit dose. One out of ten rats tested fed a diet of 5,000 mg a. i./kg

- diet was observed to be emaciated and producing few feces, suggesting possible food avoidance. It is uncertain, however, if symptoms observed in this one animal were related to the exposure to the two active ingredients. These results classify dyes c ontaining acid blue 9 and acid yellow 23 as being practically nontoxic

to birds and mammals.

In three acute aquatic tests, acute toxicity of Aquashade® is tested with bluegill sunfish, rainbow trout, and the waterflea ( Daphnia mag n a ). In all three tests, no mortality or sublethal effects were observed at the limit concentration. These results classify dyes containing acid blue 9 and acid yellow 23 as being practically nontoxic to fish and aquatic invertebrates.

12 http://www.miljoindflydelse.dk/udgiv/publications/1999/87

- 7909

- 548

- 8/pdf/87

- 7909

- 546

- 1.pdfPage 10 of 21 Table 4 . Summary of Available Data on th e Toxicity of Aquatic Dyes Containing Acid Blue 9 and Acid Yellow 23 to Terrestrial and Aquatic Organisms

Species Study Type 1 Test Material Source Results 2

Birds

Northern Bobwhite Acute Avian Oral Aquashade ® 1 433367

- 01 LD 50 > 2250 mg a.i. /kg

NOAE L = 2250 mg a.i. /kg

Mallard Acute Avian Oral Aquashade ® 433367

- 02 LD 50 > 2250 mg a.i. /kg

NOAE L = 2250 mg a.i. /kg

Northern Bobwhite Avian Dietary Aquashade ® 435034

- 03 LC 50 > 5,620 mg a.i./kg

- diet NOAEC = 5,620 mg a.i./kg

- diet

Mallard Avian Dietary Aquashade ® 435034

- 04 LC 50 > 5,620 mg a.i./kg

- diet NOAEC = 5,620 mg a.i./kg

- diet

Mammals

Laboratory rat Mammal Acute Oral Admiral® WSP 2 452811

- 01 LD 50 > 5,000 mg a.i. /kg

NOAE L < 5,000 mg a.i. /kg (1 out of 10 rats was emaciated and had few feces)

Fish

Bluegill sunfish Fish Toxicity Bluegill Aquashade ® 432975

- 02 LC 50 > 96 mg a.i. /L

NOAEC = 96 mg a.i. /L

Rainbow trout Fish Toxicity

Rainbow Trout Aquashade ® 432975

- 01 LC 50 > 96 mg a.i. /L

NOAEC = 96 mg a.i. /L

Aquatic Invertebrates

Daphnia magna Invertebrate Toxicity Aquashade ® 432975

- 03 LC 50 > 97 mg a.i. /L

NOAEC = 9 7 mg a.i. /L

1 Aquashade ® is an end

- use product containing 23. 6 3% acid blue 9 and 2.39% acid yellow 23.

2 Admiral ® WSP is an end

- use product containing 49.62% acid blue 9 and 3.05% acid yellow 23.

While data on chronic mammalian toxicity of these aquatic dye products have not been submitted to the EPA, results of chronic mammalian studies with the two dye ingredient s have been published in the open literature. Borzelleca

, et al . (1990) report on lifetime chronic toxicity/carcinogenicity studies of acid blue 9 with rats and mice. The dye used in the studies was 90% pure. In a two

- generation rat study, d ye was administered in feed to F 0 generation rats for 2 months and then in the diet to their offspring (the F 1 generation) for 30 months or until death . Test consisted of two control groups of 120 animals each and three treated group of 120 animals

administered d ietary doses of 0.1, 1.0, or 2.0% acid blue 9. There were equal numbers of males and females (60/group). There were no compound

- related effects on fertility, gestation, parturition, lactation, or pup survival. In the 2.0% group, there was a significant red uction in F 1 female body weights after week 90, and a significant reduction in survival. Final mean body weight of females was 15% less than the pooled control mean. The NOAEC for rats w as 1.0% (1 0,0 00 mg/kg

- diet ) , or 631 mg/kg

- BW/day.

In a one

- generation mouse study, dye was administered in feed to mice for 24 months or until

death . Test consisted of two control groups of 120 animals each and three treated group of 120 animals administered dietary doses of 0.5, 1.5, or 5.0% acid blue 9. There were equal n umbers of males and females (60/group). No consistent, biologically significant compound

- related adverse effects were observed at any test level. The mouse NOAEC therefore was 5.0% (50,000 mg/kg

diet ) , or 7354 mg/kg

- BW/day.Page 11 of 21 Borzelleca

, et al . (1988) repor t on two

- generation chronic toxicity/carcinogenicity studies of acid yellow 23 with rats. One ‘low

- dose” test consisted of two control groups and treated groups with 0.1, 1.0, or 2.0% acid yellow 23 . A second “high dose” test consisted of a control group a nd a 5.0% treated group

. In both tests, each g roup was composed of 60 rats per sex (120 total) in the F 0 generation, and 70 rats per sex (140 total) in the F 1 generation. No compound

- related effects were noted in any treatment group in either test. The rat NOAEC was therefore 5.0% (50,000 mg/kg

diet) or 2641 mg/kg

- BW/day.

b. Aquaculture Studies

Published studies have demonstrated that the dyes in A quashade ® do not exert any appreciable direct toxicity to aquatic plants, but merely reduce their growth through shading sunlight. A study by Manker and Martin (1984) found that hydrilla grown with Aquashade® and sodium azide did not grow significantly different th an plants growing with Aquashade® alone. Since sodium azide is known to protect cells from oxygen radicals, this experiment indicates that Aquashade® does not cause oxidative damage to plant cells through creating oxygen free radicals, which has been

propo sed as an alternative mode

- of

- action to shading. Manker and Martin (1984) also compared the growth of hydrilla growing in water containing Aquashade® with plants growing in dye

- free water, but with the light source shaded by Aquashade solutions® that were in a separate vessel placed between the light source and the vessels containing the hydrilla. This experiment found that the growth of hydrilla that were shaded by the Aquashade® solution without being in direct contact with the dyes was identical to the p lants that were grown in Aquashade® solutions. Spencer (1984) found similar results with algae growth rates , which were not reduced when grown in shallow vessels with Aquashade® concentrations less than 5 to 10 ppm. The algal cells in this experiment had d irect contact with the dyes, but because the water was shallow (less than 2 cm), there was little shading of light. In contrast, in another experiment algae was grown in dye

free water that was shaded by a 1

- meter column of water containing Aquashade®, aga in placed in a separate vessel locate between the test vessel and the light source. In this case, shading by Aquashade® concentrations of 1 and 5 ppm resulted in significantly reduced photosynthetic rates in the algae.

These experiments indicate that the aquatic dyes containing Acid Blue 9 and Acid Yellow 23 do not control aquatic plants through any direct toxicity, but merely by filtering out light used by plants for photosynthesis. The lack of a toxic mode

- of

- action at the cellular level is consistent

w ith these results of toxicity studies that show no acute toxicity in birds, mammals, fish, and daphnids when tested up to limit doses. Indeed, as shown by the experiment by Spencer (1984), the dyes will even have little effect on aquatic plankton in shallo w reaches of the water column. Effects on aquatic plants will mainly be limited to aquatic macrophages and filamentous algae growing, and aquatic plants growing in deeper water. The limitation to effects only in deeper water would explain the finding of Lu dwig (2010) that Aquashade® treatment of aquaculture ponds with an average depth of 50 cm had no significant effect on macrophyte weight and no significant effects on the growth of cyanobacteria, green algae, or euglenoid algae, nor on the zooplankton whic h feed on them. Similarly, Boyd and Noor (1982) did not find any evidence that Aquashade treatments effected the abundance of phytoplankton or the coverage of submerged aquatic plants in 1

- m deep fish ponds. Therefore, even at full treatment concentration s, these dyes are expected to have little effect on aquatic plants in shallow water such as creeks, shallow streams, or wetlands. If the dyes were inadvertently released into deep water bodies, such as lakesPage 12 of 21 or rivers, they theoretically could have adverse effects on aquatic plants, but in these cases dilution would likely reduce the dye concentrations to well below effective concentrations.

Aquatic dyes containing Acid Blue 9 and Acid Yellow 23 are used in aquaculture, and thus research has been conducted to investigate if the dyes result in any adverse effects to the fish production and the aquatic ecosystems of fish ponds. Ludwig, et al . (2010) studied the effects of Aquasha de® treatment of 0.04

- ha ponds used for fish production. The ponds were fertilized with rice bran, and stocked with 15,000 fish fingerlings. Treated ponds received two treatments each of Aquashade® at a rate of 4.8 ppm. A rate higher than the label recomme nds was used to compensate for the test ponds being shallow. The pond water was monitored for water quality and phytoplankton and zooplankton abundance was monitored for approximately 30 days. Chlorophyll a concentrations were also monitored for 4 days.

Results indicated that the Aquashade® treatments had very little effects on the fish fingerling or the ecosystem. There were no significant differences in the survival, abundance, or growth of fish between treated and control ponds. There were also no sign ificant differences in phytoplankton or zooplankton with the exception of diatoms, which were at a higher concentration in the treated ponds during the end of the study. Chlorophyll a concentrations were significantly lower in treated ponds, but there was no significant difference in respiration, gross production, or net production. There were also little differences in water quality between treated and untreated ponds. There were no significant trend for unionized ammonia and pH to be higher in the control ponds, which suggest that the moderation of algae growth may actually have some beneficial effects on water quality.

In an earlier study, Boyd and Noor (1982) evaluated the effects Aquashade® treatment of 1

- m deep, 0.04

- ha ponds stocked with fingerlings of channel catfish ( Ictalurus punctatus ). Although the study was flawed by an unrelated fish kill in one of the ponds, apparently resulting from a heavy duckweed infestation, mean fish production in the two remaining treated ponds (3,641 kg/ha) appeared to be similar to or slightly greater than mean fish production in the three control ponds (3,010 kg/ha).

Bristow, et al . (1996) compared the effects to water dyed with 2 ppm and 20 ppm Aquashade® versus clear water to the performance of cultured walleye la rvae in fish hatchery tanks. They evaluated the growth and health of the fish larvae by measurements of survival, length, terminal weight, and gas bladder inflation. At 2 ppm, which is equal to the maximum target dye concentrations for all registered produ cts, none of the performance endpoints were significantly different between the tanks with dyed water and those with clear water. Fish length was significantly reduced compared to the control in tanks with a dye concentration of 20 ppm. However, this conce ntration is 10 times greater than the maximum target concentration listed on registered products. Considering that accidental release of the dyes from treated waters would be diluted with untreated water in the receiving body, and thus be considerably less than the target concentration, this study indicates that fish larvae are not likely to be effected by registered uses of these dyes.Page 13 of 21 c. Ecological Incidents

A query of the Ecological Incident Information System (EIIS, version 2.1) in March of 201 5

identif i ed one ecological incident of adverse field effects that was associated with use of a dye containing acid blue 9 and acid yellow 23

. In October of 2010, a major bird and fish kill occurred at a country club in Lakewood County, California. An initial incid ent report from the California Department of Fish and Game (I022432

- 022) states that at least 150 dead birds (including mallards, northern shovelers, and coots) and 50 dead fish were observed at the incident site. The

10

- acre pond where the incident occurr ed had been treated with Admiral® Liquid (EPA Reg. No.

67064

- 2), which contains 15.31% acid blue 9 and 1.00% acid yellow 23. The pond had also been treated with Sonar® (EPA Reg. No. 67690

- 4, active ingredient fluridone) and a colorant product

called Black Onyx. The manufacturer of Black Onyx does not claim any pesticidal properties, and therefore the product is not regulated under FIFRA and does not have any listed active ingredients. While the cause of this incident is currently unknown, the very low toxic ity of acid blue 9 and acid yellow 23 to both birds and fish make it very unlikely that they were the cause of the observed mortality. This incident is therefore classified as “unlikely” for these two dyes.

A review of the Avian Monitoring Information System (AIMS), which is maintained by the American Bird Conservancy, did not identify any ecological incident associated with the use of either of these dyes

.

In addition to the incidents recorded in EIIS and A IMS, the Agency maintains a database which stores counts of minor incidents that pesticide registrants record in quarterly aggregated incident reports. Ecological incidents recorded in these aggregated reports include those categorized as

‘minor fish or wi ldlife’ (W

- B), ‘minor plant’ (P

- B), and ‘other non

- target’ (ONT) incidents. A

review of this database in May 201 5 identify only a single minor fish or wildlife (W

- B) and no plant or other non

- target incident associated with use of acid blue 9 and acid yel low 23. The W

- B incident (I025529

- 022) occurred in 2013 during the reporting period of March 7 to June 30, and was associated with use of Aquashade (EPA Reg. No. 33068

- 1). No other information is available

on this incident.



### 8. Exposure Pathways of Concern

Based on the use pattern of aquatic dyes containing acid blue 9 and acid yellow 23, exposure to nontarget organisms is expected to be mostly limited to organisms living in and around the water bodies which are directly treated. Aquatic organisms would be exposed from ambient exposure to the treated water. Very limited exposure may occur to aquatic organisms in nearby natural water lying downhill of the treated water bodies because of the “little outflow” that the label permits for treated water bodies, as well as by possible overflow during heavy rain events. Spray drift, runoff, and atmospheric deposition are not relevant exposure routes.

Terrestrial animals living near the treated water body may be exposed by drinking from or bathing

in the treated water body. The ecological risk assessment therefore includes an assessment of birds and mammals that are exposed via drinking water treated at the target concentration of dyes

. Because the dyes are applied directly to water and readily disperses in the water c olumn , and because both dyes have very low the volatility

, inhalation exposure is expected to be negligible.Page 14 of 21 

### 9. Status of Fate and Ecological Effects Data

Available environmental fate and ecological effects data were adequate for conducting a complete risk assessment for these aquatic dyes. Based on the curre nt use pattern and target pest ( i.e.

, certain aquatic plants), exposure to honey bees is not expected . Therefore, n o additional data are being requested at this time

.



### 10. Risk Description and Characterization

a. Summary of Previous Risk Assessment Conclusions

EFED completed an ecological risk assessment for aquatic dyes containing Acid Blue 9 and Acid

Yellow 23 in 2005 for the Reregistration Eligibility Decision (RED , USEPA, 2005 ). The registered products containing these dyes that are used for aquatic weed and algae control have not changed since that time with the exception that one product (Pond Care Algae Blocker, Reg.

No. 8709

- 6) is no longer registered. The use rates and use restrictions have also not changed. Therefore, all of the conclusions of the risk assessment conducted for the RED (D302183) still apply. The se conclusions are summarized in Section 5 of this document. In summary, the previous risk assessment concluded very low acute risk to terrestrial animals, fish, and aquatic

invertebrates. Risk to terrestrial plants was predicted to be minimal because exposure from registered aquatic uses would be minimal. Direct risk to aquatic plants was assumed.

b. Additional Information from Published Literature

Studies on the ecological effects of the use of these aquatic dyes in aquaculture systems were

discussed in Section 7 .b. The findings of these studies support the conclusions that the registered

use of these dyes will not cause any significant toxicity to fish or aquatic invertebrates . These studies also evaluated growth and developmental effects in fish , and the results indicate that the

chronic risk to fish is also low

. Finally, the studies indicate that these dyes do not exert direct toxicity to phytoplankton or submerged vegetation, but only inhibit their growth through the filtering of light. Therefore, adverse effects on aquatic plants is expected t o be limited mainly to those living in deeper water.

c. Chronic Mammalian Risk

As discussed in Section 7, published papers provide data on mammalian chronic toxicity data of Acid Blue 9 and Acid Yellow 23 that are adequate for quantitative use in risk assess ment. Because these mammalian studies were not considered in the previous risk assessment, a new

chronic mammalian risk assessment based on these data is being included in this risk assessment. These studies show that both of these dyes have very low chro nic toxicity to mammals.

Borzelleca

, et al . (1990) determined the chronic NOAEC of Acid Blue 9 to be 10,000 mg/kg

- diet

,

or 631 mg/kg

- BW/day, in the rat, and 50,000 mg/kg

- diet, or 7354 mg/kg

- BW/day , in the mouse

. Borzelleca

, et al. (1988) found that Acid Ye llow 23 to be 50,000 mg/kg

- diet, or 7354 mg/kg

BW/day, in the mouse.Page 15 of 21 A screening

- level chronic risk assessment for mammals was conducted based on the NOAEC for the most sensitive species, and the predicted exposure from mammals consuming water treated to the maximum target concentration of 2 ppm of total dye. The maximum concentration of the individual dyes is determined by multiplying the concentration of total dye (2 ppm) by the maximum proportion of the particular dye in any registered product. The max imum concentration of Acid Blue 9, which would be from use of Admiral Liquid®, would be 1.88 ppm. The maximum concentration of Acid Yellow 23, which would be from use of Aquashade®, would be 0.153 ppm.

Chronic risk to mammals was assessed in the same way that acute risk was assessed in the RED risk assessment, except using the chronic NOAELs for the toxicity endpoint. The daily dose of each dye was estimated by multiplying the volume of water expected to be consume d per day by the maximum expected dye concentration in treated water , and then dividing by the assumed body weight :

Daily Dose (mg/ kg

- day) = Daily Water Intake (L/day) × Dye Concentration (mg/L) / Body Weight (kg)

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

, the allometric equation for drinking water intake (equation 3

- 17) from the EPA Wildlife Exposure Factors Handbook (US EPA 1993) w as used:

WI (L/Day) = 0.099 (body weight in kg) 0.90

According to the labels, the maximum water concentration of total dye is 2.0 mg/L. The maximum fraction of each dye relative to total dye is 0.939 for acid blue 9 and 0.0763 for acid yellow 23. The maximum concentration of each dye is therefore 1.88 mg/L o f acid blue 9 and 0.153 mg a.i./L of acid yellow 23. Daily doses of each dye were calculated for small, medium, and large mammals according to the equation given above. T he daily doses were then divided by

the chronic r at NOAEL for each dye to calculate th e chronic risk mammalian quotients. Results of these calculations are given in Table 5

.

Table 5 . Chronic Risk Quotients for Drinking Water Exposure of Acid Dye 9 and Acid Yellow 23

Dye Assumed Body Weight (Kg) Daily Water

Intake (L/day) Daily Dose of Dye (mg a.i./kg

- day) Rat NOAEL (mg a.i./kg

- day) Chronic RQ

Acid Blue 9 0.015 0.00226 0.280 631 0.00044

0.035 0.00484 0.257 631 0.00041

1.00 0.0990 0.184 631 0.00029

Acid Yellow 23 0.015 0.00226 0.0230 2641 8.7 x 10

- 6

0.035 0.00484 0.0168 2641 6.4 x 10

- 6

1.00 0.0990 0.0151 2641 5.7 x 10

- 6

All chronic risk quotients were very small (< 0.001). Therefore, the chronic risk for mammals drinking water treated with these aquatic dyes is well below the level of concern. Combining this conclusion with the conclusions of the previously conducted risk assessment for exposure through drinking wate r, the EPA concludes no significant acute or chronic risk to terrestrial wildlife from drinking water treated with acid blue 9 and acid yellow 23

.Page 16 of 21 d. Endangered Species Assessments

In November 2013, the EPA, along with the U.S. Fish & Wildlife Service (USFWS), the National Marine Fisheries Service (NMFS) (collectively, the Services) , and the U.S. Department of Agriculture (USDA) released a summary of their joint Interim Approaches for assessing risks to listed species from pesticides. The Interim Approaches were developed jointly by the agencies in response to the National Academy of Sciences’ (NAS) recommendations and reflect a common approach to risk assessment shared by the agencies as a way of addressing scientific differences

betwe en the EPA and the Services. The NAS report 13 outlines recommendations on specific scientific and technical issues related to the development of pesticide risk assessments that EPA

and the Servi ces must conduct in connection with their obligations under the Endangered Species Act (ESA) and FIFRA.

The joint Interim Approaches were released prior to a stakeholder workshop held on November 15, 2013. In addition, the EPA presented the joint Interim Approaches at the December 2013 Pesticide Program Dialogue Committee (PPDC) and State

- FIFRA Issues Research and Evaluation Group ( SFIREG) meetings, and held a stakeholder workshop in April 2014, allowing additional opportunities for stakeholders to commen t on the Interim Approaches. As part of a phased, iterative process for developing the Interim Approaches, the agencies will also consider public comments on the Interim Approaches in connection with the development of upcoming Registration Review decision s. The details of the joint Interim Approaches are contained in the white paper “Interim Approaches for National

- Level Pesticide Endangered Species Act

Assessments Based on the Recommendations of the N ational Academy of Sciences April 2013 Report,” 14 dated November 1, 2013.

Given that the agencies are continuing to develop and work toward implementation of the Interim Approaches to assess the potential risks of pesticides to listed species and their designated critical habitat, th is ecological risk assessment for aquatic dyes containing acid blue 9 and acid yellow 23 does not contain a complete ESA analysis that includes effects determinations for specific listed species or designated critical habitat . Although EPA has not yet completed effects determinations for specific species or habitats, for this preliminary assessment EPA conducted a screening

- level

assessment for all taxa of non

- target wildlife and plants that assumes for the sake of the

assessm ent that listed species and designated critical habitats may be present in the vicinity of the application of acid blue 9 and acid yellow 23 . This screening level assessment will allow EPA to focus its future evaluations on the types of species where the p otential for effects exists once the scientific methods being developed by the agencies have been fully vetted. This screening

- level

risk assessment for acid blue 9 and acid yellow 23 indicates potential risks of direct effects to

listed aquatic plants fro m use on the registered use site. Listed species of aquatic animals and aquatic dependent wildlife may also be affected through indirect effects because of the potential

for direct effects on aquatic plants upon which such species may rely. Potential direc t effects on

listed aquatic plants from the use of acid blue 9 and acid yellow 23 may be associated with modification of Primary Constituent Elements (PCEs) of designated critical habitats, where such designations have been made. Once the agencies have ful ly vetted the scientific methods necessary to complete risk assessments for endangered and threatened (listed) species and their designated critical habitats, these methods will be applied to subsequent analyses for aquatic dyes

13 http://www.nap.edu/catalog.php?record_id=18344

14 http://www.epa.gov/espp/2013/nas.htmlPage 17 of 21 containing acid blue 9 and acid yellow 23 as part of completing this registration review.

e. Endocrine Disruptor Screening Program

Aquatic dyes containing acid blue 9 and acid yellow 23 were not included in the first group of 67

chemicals issued an order to conduct Tier 1 EDSP testing. For additional information the EDSP program, visit http://www.epa.gov/endo/

.

f. Clean Water Act Programs

Aquashade and Admiral (a cid blue 9 and acid yellow 23) have not been identified as a cause of impairment for any water bodies listed as impaired under section 303(d) of the Clean Water Act 15

. In addition, no Total Maximum Daily Loads (TMDL) have been developed for Aquashade or

Admiral 16 . More infor mation on imp aired water bodies and TMDLs can be found at the Agency’s

website 17 . The Agency invites submission of water quality data for this pesticide. To the extent possible, data should conform to the quality standards in Appendix A of the OPP Standard Operating Pro cedure: Inclusion of Impaired Water Body and Other Water Quality Data in OPP’s Registration Review Risk Assessment and Management Process 18 in order to ensure they can be used quantitatively or qualitatively in pesticide risk assessments. Aquatic benchmarks 19 have not been established for these chemicals. Any data submitted or otherwise located as part of the registration review process may be used to update aquatic life benchmarks, if applicable.

15 http://iaspub.epa.gov/tmdl_waters10/attains_nation_cy.cause_detail_303d?p_cause_group_id=885

16 http://iaspub.epa.gov/tmdl_waters10/attains_nation.tmdl_pollutant_detail?p_pollutant_group_id=885&p_pollutant_group_name=P

ESTICIDES

17 http://www.epa.gov/owow/tmdl/

18 http://www.epa.gov/oppsrrd1/registration_review/water_quality_sop.htm

19 http://www.epa.gov/oppefed1/eco risk_ders/aquatic_life_benchmark.htmPage 18 of 21 

### 11. References

Borzelleca, J.F., K. Depukat, and J.B. Halagan. 1990. Lifetime toxicity/carcinogenicity studies of FD&C blue no. 1 (brilliant blue FCF) in rats. Food Chemistry and Toxicology 2 8 ( 4 ): 221

- 234

.

Borzelleca, J.F. and J.B. Halagan. 1988. Chronic toxicity/carcino genicity studies of FD&C yellow no. 5 (tartrazine) in rats. Food Chemistry and Toxicology 26(3):179

- 187.

Boyd, C.E. and H. Noor. 1982. Aquashade® treatment of channel catfish ponds. North American Journal of Fisheries Management 2(2):193

- 196.

Bristow, B.T., R.C. Summerfelt, and R.D. Clayton. 1996. Comparative performance of intensively cultured larval walleye in clear, turbid, and colored water. The Progressive

Fish

- Culturist 58(1):1

- 10.

Flury M. and H. Flühler. 1994. Brilliant Blue FCF as a dye trace r for solute transport studies

– A toxicological Overview. J. of Environ. Qual. 23:1108

- 1112

Lonza Corporation, 2015. Colorants: Aquashade®. http://www.lonza.com/products

services/water

- treatment/non

- residential

- water/surface

- water/colorants.aspx

Ludwig , G.M., P. Perschbacher, and R. Edziyie. 2010. The e ffect of the d ye Aquashade ® on w ater

q uality, p hytoplankton, z ooplankton, and s unshine b ass, Morone chrysops × M. saxatilis

, f ingerling p roduction in f ertilized c ulture p onds . Journal of the World Aquaculture Society 41(S1): 40

- 48.

Manker, D.C. and D.F. Martin. 1984 . Investigation of t wo p ossible m odes of a ction of the i nert
d ye Aquashade ® on h ydrilla

. J. Environ. Sci. Health , A19(6):725

- 733.

Marmion D.M. (1991) Handbook of U.S. Colorants: Foods, Drugs, and Cosmetics and Medical Devices. 3rd Ed. New York,John Wiley & Sons, Inc.

MITI (1992). Biodegradation and bioaccumulation

- data of existing chemicals based on the CSCL Japan. Chemicals Inspecting & Testing Institute, Ministry of International Trade & Industry, Japan.

Pagga, U., and D. Brown. 1986. The degradation of dyestuffs: I!. Behaviour of dyestuff in aerobic biodegradation tests. Chemosphere 15:479

- 491.

Porter, J.J. 1973. The stability of acid, basic, and direct dyes to light and water. Text. Res. J.

43:735

- 744.

Spencer, D.F. 1984. Influence of aquashade on growth, photosynthesis, and phosphorus uptake of microalgae. J. Aquat. Plant Manage. 22: 80

- 84.Page 19 of 21 USEPA, 1993. Wildlife Exposure Factor Handbook . Office of Research and Development, U

. S

. Environmental Protection Agency. Washington, DC. EPA/600/R

- 93/187a

USEPA, 2005. Reregistration Eligibility Decision for Erioglaucine and Tartrazine (Aquashade*).

Case No. 4010. September 23, 2005. U.S. Environmental Protection Agency. Washington,

DC.

White, Marla P., Hippensteel, Timothy and Lembi, Carole A. 1975. Evaluation of a water

- soluble dye for aquatic weed control (abstract). Proceedings of the North Central Weed Control

Annual Conference.vol. 30.

Zollinger, H. 1991. Color chemistry. Syntheses, properties and applications of organic dyes and pigments. 2nd ed. VCH Verlagsgesellschaft, Weinheim.20 

### Appendix A

OPPIN Bibliography

Ecotoxicity



### 71

- 1 Avian Single

- Dose Oral Toxicity

MRID Citation Reference

40731 Verlangieri, A.J.; Shapiro, R.; Stern, A. (1979) Evaluation of the Acute Oral Toxicity in Mallard Ducks of: Aquashade

-- Aquatic Plant Growth Control: Report No. T

- 694. (Unpublished study received Feb 27, 1980 under

33068

- 1; prepared by Nutrition International, Inc., submitted by Aquashade, Inc., Dobbs Ferry, N.Y.; CDL:241891

- A)

43336701 Campbell, S.; Beavers, J. (1994) Aquashade: An Acute Oral Toxicity Study with the Northern Bobwhite: Lab Project Number: 196/103A. Unpublish ed study prepared by Wildlife International Ltd. 24 p.

43336702 Campbell, S.; Beavers, J. (1994) Aquashade: An Acute Oral Toxicity Study with the Mallard: Lab Project Number: 196/104. Unpublished study prepared by Wildlife International Ltd. 23 p.

4519 2101 Pedersen, C. (2000) Acute Avian Oral Toxicity (LD50) Study with Admiral WSP in Bobwhite Quail: Lab Project Number: 169

- 001

- 03. Unpublished study prepared by Bio

- Life Associates, Ltd. 38 p. {OPPTS 850.2100}.



### 71

- 2 Avian Dietary Toxicity

MRID Citation Reference

43503403 Campbell, S.; Beavers, J. (1994) Aquashade: A Dietary LC50 Study With the Northern Bobwhite: Lab Project Number: 196

- 101. Unpublished study prepared by Wildlife Int'l, Ltd. 36 p.

43503404 Campbell, S.; Beavers, J. (1994) Aquashade: A Dietary LC50 Study With the Mallard: Lab Project Number: 196

- 102. Unpublished study prepared by Wildlife Int'l, Ltd. 35 p.

45200901 Pedersen, C. (2000) Acute Avian Dietary Toxicity (LC50) Study with Admiral

W SP in Bobwhite Quail: Lab Project Number: 169

- 002

- 01. Unpublished study prepared by Bio

- Life Associates, Ltd. 69 p. {OPPTS 850.2200}



### 72

- 1 Acute Toxicity to Freshwater Fish

MRID Citation Reference21 43297501 Graves, W.; Swigert, J. (1994) Aquashade: A 96

- Hour Static Acute Toxicity Test with the Rainbow Trout (Oncorhynchus mykiss): Final Report: Lab Project Number: 196A/107A. Unpublished study prepared by Wildlife International Ltd. 39 p.

43297502 Graves, W.; Swigert, J. (1994) Aquashade: A 96

- Hour Static Acute Toxicity Test with the Bluegill (Lepomis macrochirus): Final Report: Lab Project Number: 196A/108. Unpublished study prepared by Wildlife International Ltd. 38 p.

44887610 McElwee, C. (1992 ) Lake Colorant WSP: Acute Toxicity to Bluegill Sunfish, Lepomis macrochirus, Under Static Test Conditions: Lab Project Number: J9203007B. Unpublished study prepared by Toxikon Corp. 22

p.

44887611 McElwee, C. (1992) Lake Colorant WSP: Acute Toxicity to Rainbow Trout, Oncorhynchus mykiss, Under Static Test Conditions: Lab Project Number: J9203007C. Unpublished study prepared by Toxikon Corp. 21

p.



### 72

- 2 Acute Toxicity to Freshwater Invertebrates

MRID Citation Reference

43297503 Graves, W.; Swigert, J. (1994) Aquashade: A 48

- Hour Static Acute Toxicity Test with the Cladoceran (Daphnia magna): Final Report: Lab Project Number: 196A/109. Unpublished study prepared by Wildlife International Ltd. 38 p.

44887612 McElwee, C.; Lintott, D. (1992) Lake Colorant WSP: Acute Toxicity to the Water Flea, Daphnia magna Under Static Test Conditions: Lab Project Number: J9203007A. Unpublished study prepared by Toxikon Corp. 22 p.

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.

P ublished data on the chemistry and behavior of dyes in aquatic systems was used to supplement

the environmen tal fate assessment. Most of the data come 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.