CODING FORMS FOR SRC INDEXING Microfiche No. OTS057351.5

8697000061.0

02/1.4/95 80

ANALYTICAL BIO-CHEM LABS INC

CHRONIC TOXICITY OF NONYLPHENOL TO CERIODAPHNIA DUBIA WITH COVER LETTER DATED 02/28/1997
Chemical NONYLPHENOL (CAS# 84852-15-3)**~.** c:::J:!.c:he So. _ti) 

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### u.s No.**~lO~** 24CHEMICAL MANUFACTURERS ASSOCIATION
VIA MESSENGER February28.1997 TSCADocumentProcessing Center ([5-7407) Office ofPollutionPreventionandToxies U.S. EnvironmentalProtectionAgency 401 M Street, SW Washington,DC 20460 Attention: TSCA Section 8(d) HealthAndSafetyReporting GerryBrown; IMD,EOC Programs 61 

### FR 7421; February28, 1996 61 

### FR55874; October29, 1997 DearSirorMadam: InconnectionwiththeTSCA Section8(d) Healthand SafetyReportingRules for certainalkylphenols (APs) andalkylphenolethoxyIates (APEs) noted above.the CMA Alkylphenols andEthoxyIates Panelsubmits thefinalreports ona numberofstudies on APs andAPEs thatwere sponsoredbythe PaneL This submissionisbeingmade as part ofanongoingCMA, EPA andTSCA InteragencyTestingCommitteeinformation exchangeprogram. Eachfinal studyreporthasbeenassigned a DocumentControl Number 

### <DCN), asprovidedby the EPA. This letteralso lists severalongoingstudies thatarebeingconduCtedfor the Panel Allofthese studies are identifiedbelow. Themembers ofthe CMAAlkylphenols andEthoxyIates Panelare: ExxonChemicalCompany P.O. Box563 Bldg 1 Room 3074 1900 EastLindenAvenue Linden.

### NJ 07036 GE SpecialtyChemicals 501 AveryStreet Parkersburg, WV 26102 

### HuntsmanCorporation 3040PostOakBlvd. Room 1846 Houston,

### TX 77056 Rhone-Poulenc Inc. CN7500 ProspectPlainsRoad Cranberry,

### NJ 08512-7500 SchenectadyInternationalInc. P.O. Box 1046 Schenectady, NY 12301 UnionCarbideCOIporation 3200 KanawhaTurnpike P.O. Box 8361 SouthCharleston,WV 25303

1300 WILSON BOULEVARD, ARLINGTON, VA 22209 • TELEPHONE 703-74

# ~-5000 • FAX 703-741 -6000

# ~~

# == ..**~.**TSCADocumentProcessingCenter February28,1997 Page2
DowChemicalCompany 2040 Building OSG2219**~dland,~I48674** Ciba-GeigyCorporation 540White Plains RD. P.O. Box 2005 Tarrytown, NY 10591-9005 DoverChemicalCorporation 3676 Davis RD. NW P.O. Box 40 Dover, OH44622-0040 DowComingCorporation**~ail** No. C03101 2200 VV. SalzburgRd. Midland,MI48686-0994 EthylCorporation 330 SouthFourthStreet Richmond, VA 23219 Etoxyl.

## c.A. Apartado3654 Caracas 1010A VENEZUELA Texas Petrochemicals Corp. 8600 ParkPlaceBlvd. Houston,TX 77017 Rohm and Haas Company Dept. ofToxicologyBldg. 727 NorristownRd. SpringHouse,PA 19477
SCJohnson& Son, Inc. 1525 Howe Street Racine,VVI53403-2799
UnionCamp Corporation ChemicalDevelopmentLab VV. Lathrop Ave.,P.O. Box2668 Savannah,GA 31402 Witco Corporation 3032 Brookfield Street Houston,

### TX 77045 MitsubishiChemicalCorp. 5-2 Marunouchu2-chome Chiyada-Ku,Tokyo JAPAN
The final reports submittedwiththis letter are: ChronicToxicityofNonylphenolto 

### Ceriodaphnia dubia; DCN86970000610 ChronicToxicity ofNPE 1.5 to 

### Ceriodaphnitl dubia; DCN86970000611 Determinationof

### theVaporPressure of4-Nonylphenol;DCN8697Q()Q()OlZ Nonylphenoland NonylphenolEthoxylates inRiverWater andBottom Sediments;DCN 86970000613 Toxicity ofNonylphenoltothe Amphipod

### Hyalella azteca (Saussure); DCN 86970000614 ToxicityofNonylphenolto the Midge 

### Chironomus tentans ;DCN86970000615TSCA DocumentProcessingCenter February28,1997 Page 3

The following are ongoinghealthand safety studies: 90-Day Dietary Studyin Rats Administeredpara-NonylphenoL UterineWeightAssay ofp-NonyLphenol (NP) Administered Orallyto OvariectomizedSprague DawleyRats UterineWeight Assay ofp-OctylphenolEthoxlate-5 (OPE-5) Administered Orally to OvariectomizedSprague Dawley Rats Aerobic RiverDie Awayof "c-NonylphenolEthoxylate StandardMethodfor Biodegrationbya Shake-FlaskDie-AwayMethod Nonylphenol: Metabolism,Dispositionand Pharmacokinetics Please contactmeat 703/741-5615 ifyouhave any questions. Sincerely,

# ~tkr;~ Kathryn t
.sica Manager Alkylphenols & Ethoxylates Panel Enclosures7200£ ABCLarre. Cobmrbitz, MO 65202 Tel: 3141474-8579 Far314/443·9033 86978888618



### SPONSOR Chemical Manufacrurers Association 2501 M Street NW Washington, D.C. 20037 

### STUDy TITLE Chronic Toxicity ofNonylphenol to Ceriodaphnia dubia DATA REOUIREMENT 

## u.s. EPA-TSCA 40 CFR, Part 792 Guideline 797.1300 

### AumOR Dorothy 

### E. England, M.S. Biologist IDStudy Director
STUDY COMPLETED ON February 14, 1995 PERFORMING LABORATORY ABC Laboratories, Inc. Environmental Toxicology Division 7200 East 

### ABC Lane 

### Columbia, Missouri 65202
ABC LABORATORY PROJECT ID Final Report #41756 Page 1 of409 

## Working for YouSTIJDY COMPUANCE STATEMENT Study Compliance Statement for ABC Laboratories' Final Repon #41756, entitled "Chronic Toxicity ofNonylphenol to Ceriodophnia dubia, n for the Chemical Manufacrurers Association (CMA), Washington, DC: ABC's study directorherein confirms thatthe study wasconducted incompliancewith theTSCA Good laboratory Practice Standards (40 CPR, Part 792). All original raw data were provided to CMA along with thefinal 

### repon**an~** 

### a copy was retained by ABC laboratories, Inc.

r, r**~** 

### I: 'r 

# 'Ui --rf /iJ',' / j\

### ~-.u.-~,**~Cn** 

### (.' '" -1 Vu

. Dorothy C,**~gland, ~iS,**  ABC laboratories' Study Director

Submitter**/·/~=JJ** 

### 7;- Date

Date

ABC LABS #41756-2

## OUALI1Y ASSURANCE STATEMENT ABC Laboratories' Quality Assurance Unit reviewed Study #41756, "Chronic Toxicity of Nonylphenolto Ceriodaphnia dubia," fortheChemicalManufacturersAssociation, Washington, DC. The following inspections/audits were conducted on this study. Date of Inmection

06-14-94

06-24-94 08-25-94 02-06-95 Phase Inswcted Preparation ofPrimary Standard, Working Standard, and Definitive Test Concentrations O-Hour H 20 Chemistry Draft Report and Raw Data Final Report Date Reponed to Study Director 06-14-94

06-24-94 08-25-94 02-06-95 Date Reported To Management 01-25-95

01-25-95 08-31-94 02-13-95 The undersigned conducted the fmal report and raw data audit. This audit indicated that the report is an accurate reflection of the study as it was conducted by ABC LaboratOries, Inc.

L. Me· da Anielewskr-Beeler .- Date Quality Assurance Officer

## n

ABC LABS #41756-3SIGNATIJRE PAGE Submitted 

### By:

Prepared 

### By:

Approved By:

:amk ABC Laborntories, 

### Inc. 7200 E. ABC Lane Columbia, Missouri 65202 (314) 474-8579



### Dorothy C. England,**~.S.** Biologist WSrudy Director

L. Melinda':Anielewski-Beeler Quality 

### Assurance Officer 

## n

JoniE. Rhodes, M.S. 

### Team Leader, 

### Environmental Toxicology

William A. McAllister 

### Manager, Environmental Toxicology 

### Date



## -)=.:

- 1_, -"i'=, 

### Date

Date

Date

ABC LABS #41756-4TABLE OF CONTENTS Page No. TITI.EPAGE 1 STUDY COMPUANCE STATEMENT.
. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 2 QUALITY ASSURANCE STATEMENT.
. . . . . . . . . . . . . . . . . . . . . . . . . . .. 3 SIGNATURE PAGE
......................................... 4 TABLE OF**CO~** . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 5 

### UST OF TABLES
.......................................... 6 SUMMARYIINTRODUCTION
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 7 l\1E1HODS AND MA1"ERIALS

.................................. 8 I. 

### Test Compound.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 8 

## n. Test Organisms
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . .. 

### 8 

## m. Test Method
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 9 A. 

### Biological
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ., 9 

### B. Chemical and Physical
... . . . . . . . . . . . . . . . . . . . . . . .. 9 IV. 

### Statistics........................................ 10 V. Analytical.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 10 A. 

### Preparation of Stod. Solutions
...................... 10 

### B. Method 

### ofAnalysis
. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 10 1. HPLC 

### Conditions
. . . . . . . . . . . . . . . . . . . . . . . . .. 11 2. 

### Calculations.
. . . . . . . . . . . . . . . . . . . . . . . . . . .. 11 DISCUSSION AND RESULTS.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 12 CONCLUSIONS.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 13 R:EFEREN"CES
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 22 APPENDICES

- RAW DATA
................................... 23 PROJECT PERSQNNEL
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 24

ABC LABS #41756-5UST OF TABLES 

### Table No. Page No. I 

### Chemical Characteristics of Aged Hard 

### Blended Water Used by ABC Laboratories" Environmental Toxicology Division.
. . . . . . . . . .. 15 

### IT Analytical Measurement ofNonylpbenol in, 

### Test Solutions During tbe 7-Day Exposure with CeriodLlphnia dubia
................. 

### 16 ill 

### Fortifications and Recoveries of Nonylpbenol Method Extension and Quality Control Samples During the 7-Day Exposure with CeriodLlpluzia dubia
................................. 17 

### IV Summary of Survival of Brood Ceriodaphnia dubia 

### During the 7-Day Exposure to Nonylpbenol
.............. "
. . . . . . . . . .. 18 V Summary of Reproduction for CeriodLlphnia dubia 

### During the 7-Day Exposure to Nonylpbenol
. . . . . . . . . . . . . . . . . . . . . . . . .. 19 VI Ranges For Water Quality Measurements During the 7-Day Exposure of Ceriodaphnio dubia to 

### Nonylpbenol
... "
. . . . . . . . . . .. 20 Vll Alkalinity, 

### Hardness, and Conductivity Measurements During the 7-Day Exposure of Ceriodaphnia dubia 

### to Nonylpbenol ".'."'
.. ' 21

ABC LABS #41756-6SUMMARYnNTRODUCTION Subject: "Chronic Toxicity of Nonylphenol to Ceriodaphnia dubia", ABC Laboratories' Report Number 41756. Sponsor. Chemical Manufacturers Association (CMA) Testing Facility: ABC Laboratories, Inc. Environmental Toxicology Division 7200 

### E. ABC Lane Columbia. Missouri 65202 Location of Original Raw Data and Final Report: Chemical Manufacturers Association 2501 M Street NW Washington, D.C. 20037 Test Material: Nonylphenol (High Purity); CAS #84852-15-3*; Batch No. IP890218; Date of Manufacture: November 29, 1989; >95% para-nonylphenol; Source: Schenectady International. Inc. (fonnerly Schenectady Chemicals, Inc.) Nominal Test Concentrations: Control, Acetone Vehicle Blank, 3.0, 10, 30, 100, 300, and 1000 p.g/L. Day O-I Mean Measured Concentrations: Control, Acetone VehicleBlank, 2.89, 10.4,25.9, 88.7,202, and 377 p.g/L. Dilution Water. Hardness 152-158 mg/L as 

## Caco3 , Alkalinity 164-170 mg/L as 

## Caco3 , 

### pH 8.3-8.5, and Conductivity 320-340 #Lmbos/cm Experimental Test Dates: Initiation

- June 24, 1994 Termination

- July 1, 1994 Length of Study: 7-Days Results: The 96-hourLC

### so and EC

### so were calculated at 276 p.g/L (95

### % C.1. 202-377 ",gIL) and 69.0 p.g/L (95% C.I. 44.3-110 p.gIL), respectively. The 7-day LC

### so was calculated at 258 p.glL (95 % C.1. 10.4-377 p.g/L) andthe7-day EC

### so was calculated at 99.2 p.gIL (95% C.1. 64.0-170 p.g/L) based on total adverse effects. Mortality 7-Day NOEC = 202 p.g/L 7-Day LOEC 

### = 377 p.g/L 7-Day CV = 276 ,...g/L Reoroduction 7-Day NOEC 

## = 88.7 p.gIL 7-Day LOEC 

### = 202 p.gIL 7-Day RV = 134 ",gIL Test Species: Ceriodaphnia dubia neonates 

## « 24-bours old) Source of Organisms: ABC Laboratories in-bouse cnlmre
ABC LABS #41756-7MEIHODS AND MATERIALS The static Ceriodaphniadubia bioassay was conductedfollowing theprocedures outlined inABC Laboratories' protocol 8606. The study was authorized by a representative of the Chemical Manufacturers Association on M:1Y 26, 1994 and by ABC Study Director on May 31. 1994. Thetest methods used arebasically those described inMethodsforMeasuring rheAcure Toxicity of EJIluems andReceiving Waters 10 Freshwater andMarine Organisms (1), SraruiardMethods for rhe Examinarion ofWater and Wastewater (2), and Methodsfor Acure Toxicity Tesrs with Fish. Macroinvenebrates. andAmphibians (3). I. Test Compound NOf.lylphenol. identified as batch IP890218, CAS #84852-15-3*, was received from Enviro Systems, Inc., via Exxon Biomedical, on December 3, 1992. The source of the material was Schenectady Chemicals, Inc. (Schenectady International. Inc.). Purity was listed as >95% para-nonylphenol: however, at the study sponsor's request, no correction was made forpurity. This sample was in the form of a clear viscous liquid. Upon receipt, this sample was assigned ABC laboratories' reference #TS-6181 and stored at room temperature. 

## n. Test Organisms Test specimens of Ceriodaphnia dubia were obtained from an in-house daphnid culture at ABC laboratories. The primary culture was obtained fI"('m Aquatic Biosystems, Inc., on July 27, 1990, and are maintained "in-house" at ABC laboratories. The organisms were identified to species by the supplier as Ceriodaphnia dubia. All daphnids were cultured and tested in a temperature controlled area at 25 

## ± 2°C. The lighting was maintained at 40-70 footcandles on a 16-hour daylight photoperiod, with 30 minute dawn and dusk transition periods. The daphnid cultures were maintained in 2-L glass containers. Dapbnids were cultured in hard blended water (Table I). Hard blended water is a cOmbination ofwell water and reverse-osmosis water blended to a hardness of 130-160 mg/L as CaC03. During the holding period, the daphnids were fed a suspension ofat leastonealgae speciesculturedatABC laboratories: Selenasrrum capriCOmurUTn, Ankisrrodesmusfalcatusandlor Chlamydomonasreinhardrii. Alongwiththealgae, the daphnids werefed a supplement consisting oftrout chow, supplied by Zeigler Brothers, Gardners, PA, and Fleischmann'sactive dry yeast. The daphnids were fed at least every three days. Only first-instar daphnids 

## « 24 hours old) were selected for testing. To provide test daphnids which were <24 hours old, the adult daphnids were transferred and isolated the previous day. All test daphnids were provided by ABC culture CD-39-AQT on June 24, 1994. The adults from this culture had been cultured for 

### 5 days and were considered acceptable with no signs of stress, disease, or physical 

### damage. Since the

ABC LABS #41756-8

### culruring and testing parameters oftemperature, dilution water and lighting were the same. no acclimation period was necessary. 

## m. Test Method Definitive nominal test concentrations were set by the study sponsor in a geometric series ranging from 0.0030 to LO 

### mg/L (3.0 to 1000 JLg/L). 

### Six concentrations containing ten replicates each of the test compound, acetone vehicle blank, and ABC Laboratories' hard blended water contrOl were tested. The static Ceriodaphnia dubia 

### bioassay was conducted in 50-mL pyrex glass beakers containing 30 mL of test solution. The cups were randomized in styrofoam test boards held in a 25 

## ± 2DC temperarure-controlled waterbath. The lighting was maintained at 66-78 footcandles on a 16-hour daylight photoperiod with 30-minute simulated dawn and dusk periods. Test solutions were prepared daily in l-L volumetrics and brought to test temperarure in a 25°C waterbath. Fresh solutions were added to labeled test cups and dapbnids were transferred from old to new solutions using a smooth-tipped plastic disposable pipet. All cups with new solution received 0.1 mL of algae (3.0 x107 cells/mL) plus 0.1 mL of yeast/cereal leaveslftsh food (yCT, 1.9 mg/mL). 

### for a fmal concentration of 2.0 x lOS algal cells/mL and 0.01 mg/mL 

### YCT suspended solids per cup. After water chemistry measurements were made on the "new" solutions, the remaining test solution was added to a 500-mL glass beaker and placed in the test waterbath. This solution was used the following day for water chemistry meas...rements of "old" solutions. A. Biological One Ceriodapluzia dubia (first 

### instar less than 24 hours old) was added to each replicate. Dapbnids were impartially added to test solutions as soon as test solutions were brought to test temperarure. All concentrations were observed daily for production of instar, mortality, "and other abnormal effects such as surfacing, paleness, small size, daphnids trailing: extraneous material, quiescence, anddapbnidstending tothe bottom ofthe test chambers (Appendix I.B.3.). The number cfneonates produced was recorded daily beginning with the first day ofreproduction. B. 

### Chemical and Physical Waterchemistry measurements oftemperature, dissolved oxygen, pH, and conductivity were performed daily on contrOls, vehicle blanks, and low, mid, and high test levels of the renewal (new) solutions and on reserve samples of the 24-hour old test solutions. Alkalinity and hardness measurements were made on the control and high test solutions of both old and new solutions.

ABC LABS #41756-9

### IV. Statistics Statistical analysis ofthe Ceriodaphnia dubia 

### survival data was performed using a computerized LCso/ECso program developed by Stephan et al. (4,5). This program calculated the LCsofECso statistic and its 95-percent confidence limits using the binomial, moving average, and probit tests, if

### data permitted. Three 

### different methods ofanalyzing the data wereused sinceno onemethod ofanalysis is appropriate for all 

### possible sets ofdata that may be obtained. The method of calculation selected for presentation in this report was that which gave the narrowest confidence limits for the LCsofECso (3, 4) although all three 

### .Ilodels are valid. The NOEC (no-observed effect concentration) and LOEC (lowest-observed effect concentration) endpoints weredetermined using SAS (6). Thecontrol and vehicle blank treatments were compared using chi-square and Fisher's exact test to determine suitability for pooling. Levene's test for homogeneity ofvariance was used to evaluate the necessity of data transformation using arcsine/square root transformation. A one-way analysis of variance (ANOVA) was performed to determine if 

### statistical differences existed between mean survival values among the treatment levels, followed by a Dunnett's test to compare mean survival values ofeach concentration with that Of

### the mean control values. Reproduction data, including timeto first brood and young peradult reproduction day (yAD), were analyzed in a similar manner, except that no transformation of data was performed. V. Analytical A. Preparation of Stock Solutions Primary standard solutions of NP at concentrations of 1.82 and 1.73 mg/mL were prepared in methylene chloride and acetone on May 12, 1994, and June 14, 1994, respectively. Subsequent dilutions of the stock solutions were prepared in 

### acetone for use as spiking 

### solutions and in hexa'le for use as HPLC reference standards. All standards were refrigerated when not in use; all 

### standard preparations and dilutions were recorded. B. Method ofAnalysis The measured concentrations ofNP in test water were determined from samples ofeach test level, control, vehicle blank, and NP quality control collected on Day 0 and 1ofthetest. 

### Test levels 2 and 5, control, and NP quality control were measured on Days 3, 4, 6, and 7. Concentrations were measured through the use of high perfonnance liquid chromatography (HPLC). klalysis was accomplished based 

### on modifications ofa methodprovided by CMA. This 

### method was validated for the reoovery ofNP in test 

### waterpriorto the initiatioc ofthe toxicity ABC LABS #41756-10test. Theresults ofthe method validation werepresented to CMA in ABC Report #40597. The lower range ofthe method validation was extended to 2.62 J.LgfL by preparation of duplicate samples at that concentration before initiation ofthis toxicity test. Preparation ofthe test water S31.o;;l",c; for the determination ofNP during the test was performed in the following manner: At each sampling period, 20-mL aliguOts of test water were collected in duplicate with a Class A pipetfrom each replicate and composited in a 75- mL culture tube for 

### a total volume of 40 mL. At this time, the quality control samples were fortified with NP at concentrations of 2.76 or 

### 1080 J.Lg/L (PPb). All samples were extracted with 10 mL of hexane by shaking for approximately 2 minutes. The phases were allowed to separate and the upper organicphase was drawn offusing a Pasteur pipet and placed into 

### a 15-mL culture tube. Samples were diluted in hexane such that the final NP concentrations would fall within tbe range of the analytical standards. 1. HPLC Conditions The samples were analyzed for NP by HPLC using a fluorescence detector. The chromatogrnphic data for NP were collected and stored using a DEC MicroV AX. 3800 equipped with VAX. 

### MULTICHROM software. 

### The operating 

### parameters were as follows: Column: Baker CN 250 mm x 4.6 mm x 5 micron Mobile Phase: Hexane:isopropanol (75:25), isocratic Detector: Fluorescence Excitation: 229 

### run Emission: 310 om Flow Rate: 1.0 mIJmin 

### Injection Volume: 100 J.LL Retention Time:

- 5.5 min 2. Calculations CalcuI.3.tions of NP concentrations were performed by the external standard analysis function of a DEC MicroVax 3800 equipped with VAX 

### MULTICHROM software.

ABC LABS #41756-11

### Theconcentration ofNP in

### each samplewas detennined directly from the standard 

### curve by the following equation:**:g}mL** nonylphenol equivalents volzunefor ) om standard CUTII'e analysis in mL equatum
_ ng/mL (PPb) ( ) - nonylphenol

### samp:~ An 

### example calculation for Day 0, Level 3 follows: Peak 

### Area: 94,464 p.Vs Standard 

### Curve: y = 

### 13,503.4 x - 44,577.10 where y is peak area 

### units and x is 

### ng/mL NP 

### ng/mL equivalents from standard 

### curve: 10.297 ng/mL NP

(10.297 ng/mL) (100 nIL)

_ 25.7 ngJmL (40 mL)
- nonylphenol 

### Recovery ofLevel 3: (Values rounded for calculation) 25.7 X 

### 100 

## = 86%



### 30 DISCUSSION AND RESULTS 

### Prior to the initiation ofthe test, a method validation was conducted to detennine the recovery to NP in 

### aquatic test 

### water. Fortification concentrations ranged from 5.70to 1,980 p.gfL (PPb) and yielded an average recovery of 105 

### ± 7.6%. The lower range of the vaIiaation was extended to 

### 2.62 p.gIL prior to initiating this toxicity test. Duplicate samples were prepared yielding recoveries of 2.63 and 2.78 p.gfL for 100 and 106% of the nominal fortification concentration. The 7-day chronic static renewal 

### CeriodapJznia dubia 

### study with nonylpbenol was conducted from June 24, 1994, to July 1, 1994. Analytical results are presented in Table 

## n. Mean measured concentrations of NP for days 0 and 1 were 2.89, 10.4, 25.9, 88.7, 202, and 377 p.g/L. 

### These values represent 96, 104, 86, 89, 67, and 38% ofthe nominal concentrations of 3.0, 10, 30, 100, 300, and 1000 ILgfL, 

### respectively. All test 

### results and statistical calculations are based on the day 0 and day 1 mean measured values. Mean measured concentrations for days 3-4 were 10.7 and 269 p.g/L 

### and days 6-7 were 11.6 and 296 ILgfL for test 

### levels with nominal concentrations of 10 and 300 lLgfL, respectively. These values represent 107 and 90% ofnominal test concentrations for days 3 and 4 and 116 and 99% ofnominal for days 6 and 7. Based 

### on these results, 

### NP appears to be 

### stable in 

### the test 

### system. Low recovery in 

### the ABC LABS #41756-121000 Jl.g/L test level was considered to be a result of reduced solubility in hard blended test water and should not affect the results of this study since all calculations were based on the measured test concentrations. Theprimary stock solution prepared and sampled on day 0 had a measured concentration 11.9 mg/mL, representing 119% of the nominal concentration of 10 mg/mL. Table 

## m lists fortification and recovery results of the method extension and quality control samples analyzed wjth each set ofsamples. These samples were analyzed over a nominal range of2.62 to 1080 Jl.g/L. Measured concentrations yielded recoveries averaging 95 

### ± 13% ofthe nominal fortification concentrations. TableIV summarizes the survivalofbrood Ceriodaphnia

### dubia duringthe7-day chronic survival and reproduction test with nonylphenol. Controls and vehicle blanks were not statistically different and were pooled for comparison with nonylphenol treatments. Pooled controls had 85 % survival forthe7-day exposure. Survival inthe2.89to 202 Jl.g/L treatments werebetween 80 and 100%, with no concentration response observed in this range. There was a sharp decrease in survival between 202 Jl.g/L (90%) and 377 Jl.glL (0%). Dapbnids in the 202 Jl.g/L treatment showed a statistically signilicant difference from pooled controls with respect to reproduction, producing fewer young per day per adult and exhibiting a longer time to first brood production (Table V). The high level, 1000 Jl.g!L, had no neonate production since there was 100% mortality on day 1 at this concentration. The 96-hour LC

### so and 

### EC

## so values were calculated at 276 Jl.g/L (95 % C.1. 202-377 Jl.g/L) and 69.0Jl.glL (95 % C.1. 44.3-110 Jl.glL), respectively. The7-day LC 

## so was calculated at258 Jl.glL (95 

### % C.I. 10.4-377 Jl.glL) and the 7-day 

### EC

## so was calculated at 99.2 Jl.glL (95% C.1. 64.0-170 Jl.g/L) basal on total adverse effects. Table VI summarizes the water quality parameters measured during the test. Temperatures ranged from 24 to 25°C. Dissolved oxygen concentrations ranged between 7.0 and 7.8 mg/L for the new solutions and from 6.4 to 7.9 mglL for the old solutions. DO values for the test solutions represented approximately 79-98% saturation at 24°C (corrected for local altitudinal pressure at ABC Labs) and were considered adequate for testing. The pH values ranged from 8.3-8.6 for the new solutions and 8.3 to 8.6 for the old solutions. Conductivity ranges were 280-320 Jl.mhoslcm for new solutions, 3()()"360 Jl.mhoslcm for old solutions. Table V summarizes ranges in alkalinity and hardness values for old and new test solutions as well as alkalinity, hardness and conductivity of dilution water used to prepare the test solutions. Alkalinity and hardness ranged from 152 to 172 mg/Land from 144 to 170 mglL, respectively, as CaC03. Conductivity in dilution water ranged from 320 to 340 Jl.mhoslcm. CONCLUSIONS The NOEC (no-observed-effect concentration) for survival was established at 202 Jl.glL. The LOEC (lowest-observed effect concentration) for survival was 377 Jl.glL, the highest concentration tested. The geometric mean ofthese values or chronic value for survival (CV) (equivalent to a maximum acceptable toxicant concentration or MATC), 

### was calculated at 276 Jl.glL.
ABC LABS #41756-13Although statistically reduced survival was observed at 377 JLglL, the concentration response slope was very steep. Low analytical recoveries and the absence of analytical data for days other than 0 and 1 make prediction of the concentration response problematic. Survival does not appear to be the most sensitive indicator oftoxicant stress for CeriodapJuzia dubia exposed to nonylphenoL Based on a significant increase in timeto first brood and a significant reduction in number ofyoungperadult reproduction days, a NOEC could be stated to be 88.7 p.glL, with an LOEC of 202 JLglL. The geometric**m~** of these values or reproductive value (RV) was calculated at 134 JLglL. The RV may be a more reliable indication of the chronic effects of nonylphenol to CeriodapJuzia dubia.

ABC LABS #41756-14TABLE I Chemical Characteristics of Aged Hard Blended Water Used by ABC Laboratories' Environmental Toxicology Division Dilution Water Screena Monthly Screensb Hardness Alkalinity pH Conductivity 152-158 mg/L (as CaC03) 164-170 mg/L (as CaC03) 8.3-8.5 320-340**~oslcm** Total Organic Carbon Suspended Solids Biyearly Screensc <1.0 mg/L 0.3 mg/L

Elements Chlorinated Hydrocarbons Aluminum Arsenic Boron Cadmium Chromium Cobalt Copper Fluoride Iron Lead Mercury Nickel Selenium Silver Zinc <0.20 mg/L <0.01 mg/L

0.250 mg/L <0.005 mg/L < 0.()()1 mg/L <0.05 mg/L <0.02 mg/L

0.9 mg/L <0.1 mg/L <0.003 mgiL

0.00032 mg/L <0.05 mg/L <0.005 mg/L <0.0005 mg/L <0.04 mg/L DDE DDD DDT Dieldrin Endrin a-BHC t1-BHC -y-BHC-Lindane .6.-BHC Heptachlor Heptachlor Epoxide Aldrin Chlordane Methoxychlor Toxaphene Endosulfan Sulfate Endosulfan I Endosulfan II HCB Mirex Telodrin



# --------------------- OrganQPhosphate Miscellaneous Diazinon Methyl Parathion Ethyl Parathion Malathion Ronnel Ethion Trithion Vapona 

### <0.1 JLg/L PCBs <0.02 JLg/L COD <0.02 JLg/L Un-Ionized Ammonia <0.05 JLglL Nitrite N <0.01 JLglL Nitrate N <0.02 JLg/L Total Phosphorus as P <0.05 JLg/L Residual Chlorine

<2.0 JLg/L

a Represents the values measured on dilution water used for the test b Represents the values obtained from the screens ofJune 1994 C Represents the values obtained from the screen ofJanuary 1994 <0.01 JLg/L <0.01 Ilg/L <0.01 Ilg/L <0.01 Ilg/L <O.01 llg/L <0.01 Ilg/L <0.01 JLg/L <0.01 JLg/L <O.Olllg/L <0.01 JLg/L <0.01 JLg/L <O.01 llg/L <0.3 JLg/L <0.05 JLg/L <4.0 JLg/L 

### <0.03 Ilg/L <0.01 JLg/L <0.01 JLg/L 

### <0.01 JLg/L <0.01 JLg/L <0.01 JLg/L


### < 1.0 JLg/L <10 mg/L <0.1 mg/L <0.02 mg/L

0.17 mg/L <0.05 mg/L <0.02 mg/L

Note: Results ofthe chemical analyses supporting these data are on file at ABC Laboratories, Inc. ABC LABS #41756-15

## ~ n 

# § l 

# -**~** 

# '" 

# - 01 Test Nominal Level Cone. (llg/L)
Conlrol 0

V. 0 Blink 3.0

2 10 3 30 4 100 5 300 6 1000 Siock 10 Soln. (mg/mL) 

### TABLE II Analylical Measurement of Nonylphenol in Test Solutions

During the 7-Day Exposure with Cen'odop/mia dubio

Measured (llg/L) Measured ("giL) 

### %or 

### %of DO 01 it Nom 03 D4 it Nom lIew old new old <MQLI <MQL <MQL <MQL <MQL <MQL <MQL <MQL 2.89 

### --b 2.89 96 10.9 9.87 10.4 104 9.64 11.7 10.7 107 25.7 26.1 25.9 86 83.8 93.6 88.7 89 149 254 202 67 231 307 269 90 395 359 377 38 11.9 119

• MQL 

## =minimum quantifiable limit; low standard X dilution ractor 6.90 ng/mL X 10 mL 

## = < 1.73 mg/mL 40 mL b This sample not used due 10 poor chromatography Measured (llg/L) 

### %of D6 07 it Nom new old <MQL <MQL

10.6 12.5 11.6 116

266 326 296 99TABLEm Fortifications and Recoveries of Nonylpbenol Method Extension and Q>.JaIity Control Samples During the 7-Day Exposure with 

### Ceriodaphnia dubia Measured Fortification Level Concentration Percent Sample (;.tgfL) (;.tgfL) Recovery Method Extension Low Spike A 2.62 2.63 100 Low Spike B 2.62 2.79 106 Day 0 Low Spike 2.76 2.32a 84 High Spike A 1080 851 b 79 High Spike B 1080 1060 98 Day I Low Spike 2.76 2.60 94 High SEike 1080**91~** 85 Day 3 Low Spike 2.76 2.48 90 I?ay 4 High Spike 1080 1180 

### 109 Day 6 Low Spike 2.76 2.28a 83 Day 7 High Spike 1080 1280 119 Mean 

## ± SD

- 95 

## ± 13 a Value is a mean oftwo analyses b Value is a mean ofthree analyses

ABC LABS #41756-17

### TABLE IV Summary of Survival ofBrood Ceriodaphnia 

### dubia During the 7-Day Exposure to Nonylphenol Mean Number ofLive Brood Ceriodaphnia 

### dubia Measured Test Study Days % 

### Concentration Survival {JLgfL)a 

### 0 1 2 3 

### 4 5 

### 6 7 Per Cone. Control 10 10 

### 9 9 

### 9 9 9 

### 9 90 Vehicle Blank 10 10 10 10 10 10 9 8 80 2.89 10 10 10 10 10 10 10 10 100 10.4 10 10 10 10 10 

# Ie 10 9 90 25.9 10 10 10 10 9 8 8 8 80 88.7 10 10 10 10 10 10 10 10 100 202 10 10 10 10 10 10 10 9 90 

### 377 10 0 0 0 0 0 0 0 

### Ob

a 

### Mean ofclay 0 new solution and clay 1 24-hour old solution analyses b 

### Significantly different from pooled controls by Dunnett's one-tailed test

ABC LABS #41756-18

### TABLE V Summary of Reproduction for Ceriodapllllia dllbia 

### During the 7-Day Exposure to Nonylphenol Mean Number of Young Produced per Meas. Coneentration Test Mean # Days Young Conc.a 

### Study Day Total of Young to per Adult (l'g/L) 

### # of Produced First Reproduction 0 2 3 4 5 6 7 Young Per Day Brood Day Control 0 0 0 27 16 39 100 82 256 51.2 3.2±0.7 6.1 ± 1.5 Vehicle Blank 0 0 0 31 3 54 63 47 198 39.6 3.2±0.4 4.1 ±2.8 Pooled Controls 0 0 0 58 19 93 163 129 454 45.4b 

### 3.2±0.5 5.0±2.4

2.89 0 0 0 28 29 13 114 75 259 51.8 3.1 ±0.3 5.2± 1.6 10.4 0 0 0 34 31 30 104 79 278 55.6 3.0±0.0 5.6±2.0 25.9 0 0 0 29 22 33 100 85 269 53.8 3.0±0.0 5.7± 1.5 88.7 0 0 0 26 35 8 109 101 279 55.8 3.2±0.6 5.9±2.2 202 0 0 0 0 6 21 II 

### 24 62 15.5 4.6±0.5c 

### 2.4± 1.9c

## ~ 377 

### 0 0 0 0 0 0 0 0 0 

### n 

# § a 

### Mean ofday 0 new solution and day 124-hour old solution analyses b 

### Mean oftwo sets ofcontrols**~** c 

### Significantly different from pooled controls by Dunnett's one-tailed test



# ....**~ ~**  

## .... \0

# ~ 

# §**~** 

# - iJt

## ~ o Nominal Test Conc. Temp"

(mg/L) (0C) Control 24-25 Vehicle Blank 24-25 0.0030 24-25 0.010 0.030 24-25 0.10 O.JO 24-25 1.0 24-25 

### TABLE VI Ranges for Water Quality Measurements During the 7-Day Exposure of CeriodapJlllia dllbia 

### to Nonylphenol
New Solutions· Old Solutions· Temp." D.D.c Condo D.D.c (mg/L) pHd (/4mhos/cm)e (OC) (mg/L) pHd 7.3-7.8 8.4-8.6 300-330 24-25 7.2-7.9 8.5-8.6 290-320 7.0-7.8 8.3-8.6 24-25 6.4-7.6 8.3-8.6 7.2-7.8 8.3-8.6 280-320 24-25 6.5-7.6 8.4-8.6

7.2-7.8 8.3-8.6 300-310 24-25 6.4-7.6 8.4-8.6

7.2-7.6 8.3-8.6 JOO-320 24-25 6.7-7.4 8.4-8.5 7.6-7.8 8.3-8.4 290-300 24-25 7.2-7.3 8.5-8.6 Condo (/4mhos/cm)C

310-340
300·340 300-340
300-340

300-360 JOO-310
a 

### Water quality measurements for the new solulions were made on the parent slock solutions at each renewal. Water quality measurements for the old solutions were made on separate beakers ofthe 24-hour old test solutions. b 

### Temperature (OC)

- Monitored using a mercury thennometer; all values arc rounded to 2 significant figures C 

### Dissolved Oxygen Concentrations

- YSI Model 54 ARC Dissolved Oxygen Meter Dissolved oxygen concentrations at the test**t~mperatures** 

### of 24 and 25°C are 8. I and 7.9 mg/L, respectively (correcled for altitudinal pressure at Columbia, Missouri. d 

### pH

- Beckman ifJ34 pH Meter with a Model 39841 probe. e 

### Conductivity measured with a YSI 33 salinity/conductivity/temperature meterTABLE VII Alkalinity, Hardness, and Conductivity Measurements During the 7-Day Exposure of 

### Ceriodaphnia dubia to Nonylphenol**Alkalini~ Hardn~**  (mg/L 

## CacoJ) (mg/L 

## CacoJ) New Solutions Control 152-162 144-160 Level516c 154-162 144-154 Old Solutions Control 160-172 150-170 Level5/6c 164-170 144-160 Blended Water 164-170 152-158

a Detennined by titrimetric procedures adapted from Standard Methods b Measured using 

### a YSI Model 33 salinity/conductivity/temperature meter C Measured in the bighest concentration having live test organisms d Presented in previous table e Used to prepare new test solutions on days 0-6 Conductivityb**(~hoslcm)**

320-340

ABC LABS #41756-21REFERENCES (1) Weber, C.l. 1991. 

### MeJhodsjorMeasuring the Acure Toridry ojEjJl.uems and

### Receiving Waten to Freshwater and 

### Marine Organisms, 4th ed. U.S. Environmental Protection Agency, EPAJ600/4-851013, September 1991. 216 p. (2) American Public Health Association. 1980. 

### Standard Methodsjor the Excmlnmion of Water and 

### Wastewater. 15th ed. Washington, DC. 1134 p. (3) Committee on Methods for Toxicity Tests with Aquatic Organisms. 1975. 

### Methodsfor AcU!e Toridty Tests with Fish, Macroinvenebrates and 

### Amphibians. Environmental Protection Agency, Ecological Research Series EPA-660/3-75-009, April 1975. 61 p. (4) Stephan, C. E., K. A. Busch, R. Smith, 

### J. Burke and 

### R. W. Andrews. 1978. A Computer Program for Calculating an ECso. U. S. Environmental Protection Agency, Duluth, Minnesota, pre-publication manuscript, August, 1978. (5) Stephan, C. E. 1977. Methods for Calculating an ECso, p. 65-84. In F. L. Mayer and 

### J. L. Hamelink (eds.). Aquatic Toxicology and Hazard Evaluation. ASTM Special Technical Publication 634. ASTM. Philadelphia. (6) PC DOS SAS/STAT Release 6.04 copyright 1985, 1987, 1988 by SAS Institute Inc., Cary, NC 27512-8000, USA. (7) U.S. Environmental Protection Agency. 1989. Toxic Substances Control Act**(TSCA)~** Good Laboratory Practice Standards; Final Rule (40 CPR, 

### Pan 792). 

### Federal Regisler, Vol. 54; No. 158:34043-34050.

ABC LABS #41756-22