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) 
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CHEMICAL MANUFACTURERS ASSOCIATION 

VIA MESSENGER 

February 28.1997 

TSCA Document Processing Center ([5-7407) 

Office of Pollution Prevention and Toxies 

U.S. Environmental Protection Agency 

401 M Street, SW 

Washington, DC 20460 

Attention: TSCA Section 8(d) Health And Safety Reporting 

Gerry Brown; IMD, EOC Programs 

61 FR 7421; February 28, 1996 

61 FR 55874; October 29, 1997 

Dear Sir or Madam: 

In connection with the TSCA Section 8(d) Health and Safety Reporting Rules for 

certain alkylphenols (APs) and alkylphenol ethoxyIates (APEs) noted above. the CMA 

Alkylphenols and EthoxyIates Panel submits the final reports on a number of studies on 

APs and APEs that were sponsored by the PaneL This submission is being made as 

part of an ongoing CMA, EPA and TSCA Interagency Testing Committee information 

exchange program. Each final study report has been assigned a Document Control 

Number <DCN), as provided by the EPA. 

This letter also lists several ongoing studies that are being conduCted for the 

Panel All of these studies are identified below. 

The members of the CMA Alkylphenols and EthoxyIates Panel are: 

Exxon Chemical Company 

P.O. Box 563 Bldg 1 Room 3074 

1900 East Linden Avenue 

Linden. NJ 07036 

GE Specialty Chemicals 

501 Avery Street 

Parkersburg, WV 26102 

Huntsman Corporation 

3040 Post Oak Blvd. 

Room 1846 

Houston, TX 77056 

Rhone-Poulenc Inc. 

CN7500 

Prospect Plains Road 

Cranberry, NJ 08512-7500 

Schenectady International Inc. 

P.O. Box 1046 

Schenectady, NY 12301 

Union Carbide COIporation 

3200 Kanawha Turnpike 

P.O. Box 8361 

South Charleston, WV 25303 

1300 WILSON BOULEVARD, ARLINGTON, VA 22209 • TELEPHONE 703-74 ~-5000 • FAX 703-741 -6000 ~~ == .. ~. 
TSCA Document Processing Center 

February 28,1997 

Page 2 

Dow Chemical Company 

2040 Building 

OSG2219 

~dland,~I48674 

Ciba-Geigy Corporation 

540 White Plains RD. 

P.O. Box 2005 

Tarrytown, NY 10591-9005 

Dover Chemical Corporation 

3676 Davis RD. NW 

P.O. Box 40 

Dover, OH 44622-0040 

Dow Coming Corporation 

~ail No. C03101 

2200 VV. Salzburg Rd. 

Midland, MI 48686-0994 

Ethyl Corporation 

330 South Fourth Street 

Richmond, VA 23219 

Etoxyl. c.A. 

Apartado 3654 

Caracas 1010A 

VENEZUELA 

Texas Petrochemicals Corp. 

8600 Park Place Blvd. 

Houston, TX 77017 

Rohm and Haas Company 

Dept. of Toxicology Bldg. 

727 Norristown Rd. 

Spring House, P A 19477 

SC Johnson & Son, Inc. 

1525 Howe Street 

Racine,VVI53403-2799 

Union Camp Corporation 

Chemical Development Lab 

VV. Lathrop Ave., P.O. Box 2668 

Savannah, GA 31402 

Witco Corporation 

3032 Brookfield Street 

Houston, TX 77045 

Mitsubishi Chemical Corp. 

5-2 Marunouchu 2-chome 

Chiyada-Ku, Tokyo 

JAPAN 

The final reports submitted with this letter are: 

Chronic Toxicity of Nonylphenol to Ceriodaphnia dubia; DCN 86970000610 

Chronic Toxicity of NPE 1.5 to Ceriodaphnitl dubia; DCN 86970000611 

Determination of the Vapor Pressure of 4-Nonylphenol; DCN 8697Q()Q()OlZ 

Nonylphenol and Nonylphenol Ethoxylates in River Water and Bottom 

Sediments; DCN 86970000613 

Toxicity of Nonylphenol to the Amphipod Hyalella azteca (Saussure); DCN 

86970000614 

Toxicity of Nonylphenol to the Midge Chironomus tentans ; DCN 86970000615 
TSCA Document Processing Center 

February 28,1997 

Page 3 

The following are ongoing health and safety studies: 

90-Day Dietary Study in Rats Administered para-NonylphenoL 

Uterine Weight Assay of p-NonyLphenol (NP) Administered Orally to 

Ovariectomized Sprague Dawley Rats 

Uterine Weight Assay of p-Octylphenol Ethoxlate-5 (OPE-5) Administered 

Orally to Ovariectomized Sprague Dawley Rats 

Aerobic River Die Away of "c-Nonylphenol Ethoxylate Standard Method for 

Biodegration by a Shake-Flask Die-Away Method 

Nonylphenol: Metabolism, Disposition and Pharmacokinetics 

Please contact me at 703/741-5615 if you have any questions. 

Sincerely, 

~tkr;~ Kathryn t . sica 

Manager 

Alkylphenols & Ethoxylates Panel 

Enclosures 
7200 £ ABC Larre. Cobmrbitz, MO 65202 

Tel: 3141474-8579 Far 314/443·9033 

86978888618 

SPONSOR 

Chemical Manufacrurers Association 

2501 M Street NW 

Washington, D.C. 20037 

STUDy TITLE 

Chronic Toxicity of Nonylphenol 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 of 409 

Working for You 
STIJDY COMPUANCE STATEMENT 

Study Compliance Statement for ABC Laboratories' Final Repon #41756, entitled "Chronic 

Toxicity of Nonylphenol to Ceriodophnia dubia, n for the Chemical Manufacrurers Association 

(CMA), Washington, DC: 

ABC's study director herein confirms that the study was conducted in compliance with the TSCA 

Good laboratory Practice Standards (40 CPR, Part 792). 

All original raw data were provided to CMA along with the final 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 

Nonylphenol to Ceriodaphnia dubia," for the Chemical Manufacturers Association, 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 of Primary Standard, 

Working Standard, and 

Definitive Test Concentrations 

O-Hour H20 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 #41756-3 
SIGNATIJRE 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 #41756-4 
TABLE 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 MA 1"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 of Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 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 #41756-5 
UST 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 of Nonylpbenol 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 #41756-6 
SUMMARYnNTRODUCTION 

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 Vehicle Blank, 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-hour LCso and ECso 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 LCso was 

calculated at 258 p.glL (95 % C.1. 10.4-377 p.g/L) and the 7-day ECso 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 #41756-7 
MEIHODS AND MATERIALS 

The static Ceriodaphnia dubia bioassay was conducted following the procedures outlined in ABC 

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. 

The test methods used are basically those described in Methodsfor Measuring rhe Acure Toxicity 

of EJIluems and Receiving Waters 10 Freshwater and Marine Organisms (1), Sraruiard Methods 

for rhe Examinarion of Water and Wastewater (2), and Methods for Acure Toxicity Tesrs with 

Fish. Macroinvenebrates. and Amphibians (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 for purity. 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 

of well 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 of at 

least one algae species cultured at ABC laboratories: Selenasrrum capriCOmurUTn, 

Ankisrrodesmusfalcatus andlor Chlamydomonas reinhardrii. Along with the algae, 

the daphnids were fed a supplement consisting of trout chow, supplied by Zeigler 

Brothers, Gardners, PA, and Fleischmann's active 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 of temperature, 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 ± 2 DC 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, and dapbnids tending to the bottom of the 

test chambers (Appendix I.B.3.). The number cf neonates produced was 

recorded daily beginning with the first day of reproduction. 

B. Chemical and Physical 

Water chemistry measurements of temperature, 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 of the 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 of analyzing the data were used since no one method of analysis 

is appropriate for all possible sets of data 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 were determined using SAS (6). The control and vehicle 

blank treatments were compared using chi-square and Fisher's exact test to 

determine suitability for pooling. Levene's test for homogeneity of variance was 

used to evaluate the necessity of data transformation using arcsine/square root 

transformation. A one-way analysis of variance (ANOV A) 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 of each concentration with that Of the mean control values. Reproduction 

data, including time to first brood and young per adult 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 of Analysis 

The measured concentrations of NP in test water were determined from 

samples of each test level, control, vehicle blank, and NP quality control 

collected on Day 0 and 1 of the test. 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 of a method provided by CMA. This method was validated 

for the reoovery of NP in test water prior to the initiatioc of the toxicity 

ABC LABS #41756-10 
test. The results of the method validation were presented to CMA in ABC 

Report #40597. The lower range of the method validation was extended 

to 2.62 J.LgfL by preparation of duplicate samples at that concentration 

before initiation of this toxicity test. 

Preparation of the test water S31.o;;l",c; for the determination of NP 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 pipet from 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 organic phase was drawn off using 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 V AX. 

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 
The concentration of NP in each sample was detennined directly 

from the standard curve by the following equation: 

:g}mL nonylphenol 

equivalents volzune for ) 

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 of Level 3: (Values rounded for calculation) 

25.7 X 100 = 86% 

30 

DISCUSSION AND RESULTS 

Prior to the initiation of the test, a method validation was conducted to detennine the recovery 

to NP in aquatic test water. Fortification concentrations ranged from 5.70 to 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% of the 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% 

of nominal test concentrations for days 3 and 4 and 116 and 99 % of nominal for days 6 and 7. 

Based on these results, NP appears to be stable in the test system. Low recovery in the 

ABC LABS #41756-12 
1000 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. The primary 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 of samples. These samples were analyzed over a nominal range 

of 2.62 to 1080 Jl.g/L. Measured concentrations yielded recoveries averaging 95 ± 13 % of the 

nominal fortification concentrations. 

Table IV summarizes the survival of brood Ceriodaphnia dubia during the 7 -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 for the 7-day exposure. Survival in the 2.89 to 202 Jl.g/L treatments were between 

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 LCso and ECso values were calculated at 276 Jl.g/L (95 % C.1. 202-377 Jl.g/L) and 

69.0 Jl.glL (95 % C.1. 44.3-110 Jl.glL), respectively. The 7-day LCso was calculated at 258 Jl.glL 

(95 % C.I. 10.4-377 Jl.glL) and the 7-day ECso 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/L and 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 of these values or chronic value for survival (CV) 

(equivalent to a maximum acceptable toxicant concentration or MATC), was calculated at 

276 Jl.glL. 

ABC LABS #41756-13 
Although 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 of toxicant stress for CeriodapJuzia dubia exposed 

to nonylphenoL Based on a significant increase in time to first brood and a significant reduction 

in number of young per adult 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 #41756-14 
TABLE 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 of June 1994 

C Represents the values obtained from the screen of January 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 of the 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) 

D6 07 it 

new old 

<MQL <MQL 

10.6 12.5 11.6 

266 326 296 
TABLEm 

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 851b 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 of two analyses 

b Value is a mean of three analyses 

ABC LABS #41756-17 
TABLE IV 

Summary of Survival of Brood Ceriodaphnia dubia 

During the 7-Day Exposure to Nonylphenol 

Mean Number of Live 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 of clay 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. Coneent ration 

Test Mean # Days Young 

Conc.a Study Day Total of Young to per Adu

(l'g/L) # of Produced First Reproduct

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.

Vehicle 

Blank 0 0 0 31 3 54 63 47 198 39.6 3.2±0.4 4.1 ±2.

Pooled 

Controls 0 0 0 58 19 93 163 129 454 45.4b 3.2±0.5 5.0±2.

2.89 0 0 0 28 29 13 114 75 259 51.8 3.1 ±0.3 5.2± 1.

10.4 0 0 0 34 31 30 104 79 278 55.6 3.0±0.0 5.6±2.

25.9 0 0 0 29 22 33 100 85 269 53.8 3.0±0.0 5.7± 1.

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± 1.9

~ 377 0 0 0 0 0 0 0 0 0 n 

§ a Mean of day 0 new solution and day 1 24-hour old solution analyses 

b Mean of two sets of controls 

~ 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 (/4 mhos/c m)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 

Co

(/4mho

310

300

300

300

300

JOO

a Water quality measurements for the new solulions were made on the parent slock solutions at each renewal. Water 

measurements for the old solutions were made on separate beakers of the 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 (correc

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 meter 
TABLE 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 #41756-21 
REFERENCES 

(1) Weber, C.l. 1991. MeJhodsjor Measuring the Acure Toridry oj EjJl.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 Methods jor the Excmlnmion of 

Water and Wastewater. 15th ed. Washington, DC. 1134 p. 

(3) Committee on Methods for Toxicity Tests with Aquatic Organisms. 1975. Methods for 

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