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ACUTE AND CHRONIC TOXICITY OF NONYLPHENOL TO TEN SPECIES OF AQUATIC ORGANISMS



### by

Mr. Larry T. Brooke, Project Leader Environmental Health Laboratory Lake Superior Research Institute University of Wisconsin-Superior Superior, WI 54880



### to

Mr. Robert L. Spehar, Project Officer u.S. Environmental Protection Agency

Environmental Research Laboratory Duluth, MN 55804



### for

u.s. EPA COntract No. 68-Cl-Q034 Partial Deliverable for Work Assignment No. 2



### March24'~ ~o ~. r.

## --

,.

Although the information described inthis article has been funded wholly or inpart by the Environmental Protection Agency under contract 68-CI-0034 entitled "Technical support services forwater quality criteria and related activities," itdoes not necessarily reflect the views of the Agency anno official endorsement should be inferred.



### - --The Environmental Health Laboratory at the University of WisconsinSuperior (UW-S) conducted tests to complete the data set necessary to derive national 1~aterquality criteria for nonylphenol. The tests were the eight acute tests specified in the "Guidelines forderiving numerical national water quality criteria forthe protection of aquatic organisms and their uses" (Stephan f!tale 1985). Two acute tests with plants were also conducted. The Guidelines require three chronic tests to derive a chronic criterion. Two chronic tosts were conducted with freshwater species sensitive to nonylphenol. The third chronic must be for a saltwater species ifboth fresh and saltwater criteria ",reto be developed. Acute 96-hr flow-through tests were conducted with nonylphenol using: an annelid, Lumbriculus variegatus; a snail, Physella virgata; a dragonfly, Ophiogomphus sp.; an amphipod, Hyalella Azteca; the fathead minnow, Pimephales promelas; the bluegill, Lepomis macrochirus; rainbow trout, Oncorhynchus mykiss; and aduckweed, Lemna minor. An acute renewal test was conducted with the cladoceran, Daphnia magna and an acute static test was conducted with the green algae, Selenastrum capricornutum. Two chronic exposures were conducted with nonylphenol. They were a 21- day exposure with the cladoceran, D. magna and a 91-day test with rainbow trout. The tests were conducted according to the procedures recommended by the American Society for Testing and Materials (ASTM) when procedures existed, except the duckweed test which deviated fromASTM recommendations.



### METHODS Test Oraanisms Annelids were cultured in the UW-S culture unit (the stock culture originated from Eastman Kodak laboratories, Rochester, NY) with flowing ~aboratory water in 57-L glass chambers. Shredded unbleached paper hand towels were used as substrate to culture the annelids and they were fed a daily diet of adult brine shrimp (Artemia salinus, San Francisco Bay brand). At the start of the test, large (adult) annelids were carefully removed from 

### 2the substrate and ten individuals were randomly assigned to each chamber. Adult snails were obtained from cultures maintained at the U.S. EPA

. Environmental Research Laboratory-Duluth (ERL-D), KN, and acclimated to test conditions for seven days prior to test initiation. They were feeding on wild growths of fungi and algae on the culture chamber walls. Dragonfly nymphs were collected from feral populations "inthe Eau Claire River near Gordon, WI. They were transported to the UW-S laboratory and acclimated for seven days in large fiberglass tanks with flowing laboratory water. The detritus collected during capture of the nymphs was used as substrate and food during the acclimation period. Amphipods originated from feral populations inthe St. Louis River, Douglas ~ounty, WI. They were cultured inbattery jars containing two liters of lab water and a substrate of 100% cotton gauze. Solutions of food and water in the culture jars were renewed weekly and the amphipod populations
were culled to approximately 50 adults per jar. Twenty milliliters of YTC

(yeast-trout chow-cereal leaves) and 50mL of inoculum consisting of miscellaneous green algae from the previous culture water were added to each jar. An udditional 20mL of YTC was fed on the second and fourth day after renewal. Organisms of about 2mm total length (juveniles) were selected for the test. Fathead minnow embryos frombrood cultures maintained at UW-S were hatched and reared 25 to 35 days in flowing labwater. The frywere fed live brine shrimp nauplii (A. salinus, San Francisco Bay Brand) three times daily until 24 hr before test initiation. certified disease-free rainbow trout were acquired from two commercial suppliers. The chronic exposure was conducted with embryos supplied as ova and sperm from the Mount Lassen Trout Farms, Inc., Red Bluff, CA. Upon arrival at UW-S, 5,000 ova from three females and sperm from four males were combined in 1.0 L of water. Water hardening (imbibition of water into fertilized ova) was complete by two hr after fertilization. Embryos were immediately drawn into aglass tube, counted and placed in random order into 

### 3-- -

-- - --

incubation cups within the diluter. Fry for the acute test were acquired from Seven Pines Trout Farm, Lewis, WI., acclimated for seven days in laboratory water, 'and fed trout chow. At the beginning of the exposure, the frywere 4S days post-hatch. Bluegill juveniles (~lyr-old) were acquired from Kloubek Fish Hatchery, Amana, IA. They were acclimated in laboratory water for seven days prior to test initiation and fedtrout chow. 

### CladoceranswereacquiredfromERL-D and cultured at UW-S inhard (160- 180 mg/L as CaC03) reconstituted water (ASTM 1991a) which was renewed three times weekly (MWF). Cladocerans were fed YTC and agreen algae (So capricornu&um) on aMWF regimen. Test organisms were chosen from the parents fourth brood. The green algae inoculum was obtained from Carolina Biological ~upply, Inc., Burlington, NC. The algae was cultured in anutrient media (ASTM 1991b) aseptically prepared. Nutrient media and inoculum were placed in a 4.0 L Erlenmeyer flask, aerated, and stirred bymagnetic stirrer for 7 to 10 days. Algal cells were centrifuged to concentrate and the supernatant decanted. The concentrated algal cells were resuspended in a small volume of media solution and quantified with ahemacytometer forpreparation of the desired cell density to initiate the toxicity test. Duckweed was obtained from the ERL-D culture. Itwas acclimated and grown with nutrient media (ASTM 1991c) in a static culture chamber with weekly addition of nutrient media. Plants that were at the two frond stage of development were used to initiate the toxicity test rather than the three or four frond stage recommended byASTM. These were found to have abetter growth response.
Exposure Conditions The water supply for nearly all culturing, acclimation and tests was the municipal water supply for the city of Superior, WI. The city water was dechlorinated by passing through an activated charcoal column and by sodium 

### 4sulfite addition. The water also passed through a cation exchange resin to
remove heavy metals. The exception to the municipal water supply as a source of culture and test water, were two cladoceran tests and the green algae test which utilized deionized water with salts and nutrients incorporated. All organisms were cultured at or near the test temperatures (Table 1). Flow-through tests were conducted using proportional mini-diluter systems (Benoit, et ale 1982). All test systems delivered toxicant solutions at a 0.5 dilution factor. Organisms were exposed to five toxicant 

### concentrationsandacontrol,all induplicate. Testchambersforallflow-

. through tests, except the rainbow trout chronic test, contained 2.4 L of test solution. All surfaces exposed to test solutions were constructed of glass, plastic, or stainless steel. Tests with dragonfly, fathead minnow, bluegill, rainbow trout (acute), and duckweed, as test organisms, were conducted in these chambers. To prevent escape, snails were placed inside nylon mesh cylinders (Sx 15 cm) capped with neoprene stoppers and placed in the test chambers. Amphipods were placed in 250 mL beakers with two 1.6 cmdiameter holes cov(lredwith stainless steel mesh located on opposite sides of the beaker. Stainless steel mesh screen was used as a substrate for the amphipods. Self-starting syphons partially drained the test chambers, cyclically, which exchanged the test solution in the beakers. Animals in acute tests were not fedduring the exposure, andmortalities observed were recorded daily. Cladocerans were acut~ly exposed in 100 mL glass beakers containing 80 

### mLoftestsolution.

.

### Fourreplicateswerepreparedforthecontrolandeach of the five toxicant concentrations before adding five organisms to each test chamber. The organisms were not fed during the exposure and were observed daily for mortalities. The test was conducted in a temperature and photoperiod (16L:8D) controlled chamber. The rainbow trout chronic exposure was conducted in a flow-through diluter with two-liter exposure chambers placed in a temperature controlled water bath. Two replicates of five exposure concentrations of nonylphenol and


### 5control were used. Each replicate contained 100 freshly fertilized embryos. The embryos were placed inoscillating incubation chambers consisting of 100
, mL beakers with the bottoms removed and replaced with nylon mesh (twoper replicate with 50 embryos ineach). The incubation chambers were slowly raised and lowered to facilitate movement of test solution around the embryos. The organisms were kept inthe oscillating incubation chambers without direct lighting until swim-up of the fry occurred (approximately 45 days). Fifteen frywere randomly chosen from the two oscillating incubation chambers and released into each test chamber. At this time, photoperiod (16L:8D) was initiated and the frywere fed. Feeding consisted of a ration of trout chow three times aday. Test chambers were given equal amounts and the quantity was increased with time~ Enough food was given to have excess on the bottom of the test chambers. The chambers were siphoned clean daily. Test duration was 91 days. The cladoceran chronic test was conducted in 100 mL plastic cups (Solo Cup Co., Chicago, IL) containing 80 mL of test solution. The tests cups were pre-conditioned with test solutions for 24 hrprior to starting the test. A single cladoceran «24-hr-old) was placed ineach of ten replicate chambers for each exposure concentration and control. Five concentrations of nonylphenol and a control were tested. Test solutions were renewed every MWF for 21 days. At renewal the organisms were fed amixture of YTC andgreen algae (ASTM 1991a) and the surviving adults were transferred by pipet to the fresh solution. If neonates were present, they were counted and discarded. The duckweed test was conducted in amini-diluter that was modified to receive toxicant and nutrient solution. The toxicant had aO.S dilution factor but the nutrient solution was delivered equally to all test chambers. The nutrients were essentially the same as inthe ASTM method (ASTM 1991c), but were prepared in three stock solutions that should not be premixed. Each solution was pumped to the water supply before it entered the diluter. Addition of the nutrients slightly increased alkalinity and hardness of the test water. Fronds were counted daily. The test did not meet the ASTM


### 6.. .. -.- -------

requirements for duration or frond production, but was sufficient to produce
an effect. Also, the test lighting did not meet the ASTM minimum requirements for inten!lityor uniformity. The green algae was exposed to nonylphenol in 300 mL Erlenmeyer flasks containing 100 mL of sterile nutrient media. The test consisted of a control and seven treatment concentrations of nonylphenol, all in triplicate. A r fourth flask was added for each treatment concentration and control as a surrogate for chemical determinations to avoid removing algae during sampling. Algal cells were added to the media to yield a cell density of 2 x 10s/mLat initiation of the test. The flasks were stoppered with foam plugs and incubated on a shaker table at 120 rpm in a constant temperature room with a 16L:8D photoperiod of 218 ft-c. At test termination, the contents of each test flas]cwere filtered through arinsed, oven dried and weighed Gelman type A-E glass fiber filter (Gelman Sciences, Inc., Ann Arbor, HI). The filters
were again dried at 65 C, cooled in adesiccator and reweighed. Weight corrections were made for flocculent present in the nutrient solution. 

### Temperature,totalalkalinity,totalhardness (EDTA) 

### andpHwere determined in the test solutions (Table 1) for all tests. Inmost tests dissolved oxygen and conductivity were measured. Temperatures were measured daily in at least a low, medium and high exposure concentration. Dissolved oxygen was measured every 48 hr inthe control, low, medium and high exposure concentrations, starting at test initiation for acute tests, and at least twice aweek in chronic exposures. Total hardness, total alkalinity, pH and conductivity were determined at least once during an acute exposure andweekly for the chronic exposures incontrol, low, medium and high exposure concentrations. All nonylphenol sto~k solutions were generated with a liquid/liquid equilibrator (noorganic solvents were used). Nonylphenol with adensity less than water was added to a container of test water (ornutrient media for the green algae) where it floated on the surface. The water was stirred with a magnetic stirrer. Stock solution was withdrawn by pump near the bottom of the 

### 7flask and fresh water .wasadded to replace the solution removed bymeans of a pump activated by a solution level sensor.
Analvtical .Procedures Chemical measurements were made by High Performance Liquid Chromatography (HPLC). The HPLC system consisted of .twoWaters H-45 

### pumps, ~waters automated gradient controller, aWaters M-490 UV detector (Waters Associates, Millford, MA), aMicromeritics Model 725 autoinjector (Micromeritics. Instrument Corp., Norcross, GA) or aValco C6Wmanual injection valve (Valco Instrument Company Inc., Houston, TX) and.a Spectra-Physics SP4270 integrator (Spectra-Physics, San Jose CA). ALiChrocart 125-4 cartridge column with LiChrospher 100 RP-l8 (5~m) packing (EM Science, Cherry Hill, NJ) or a Chromasil 4.6 x 250mm C-18 with 10~ packing (American Scientific Products, McGaw Park, IL) was used for the separation. The mobile phase varied for some tests to optimize analytical peak shape. HPLC conditions varied due to availability of solvents and the need for different detection limits (Table 2). The Ilonylphenol [CASNo. 25l54-52-3} was obtained fromAldrich Chemical Company (liilwaukee,WI) as atechnical grade mixture of ring and chain isomers. The purity of the compound as 4-nonylphenol is reported to be approximately 90 percent (Bhatt et al., 1992; Naylor 1993). The analytical 

### stocksolutionwasobtainedbydissolvingthechemicalinacetonitrile (UV Grade, Am"~ricanBurdick &Jackson, Muskegon, HI). Standards were prepared from this stock by dilution first in acetonitrile (UVGrade) and then in Hilli-Q (~IilliporeCorp., Bedford, MA) water, dechlorinated lab water or hard reconstituted water, depending upon the type of test. At least four standards
were used to construct calibration curves covering the range of sample ~oncentrations being measured. Samples were taken byglass pipet from the center of the test chambers (or the center of the beakers within the chambers) atmid-depth and placed in autoinjector vials or solvent rinsed, screw cap tubes for the manually 

### 8injec~ed tes~s. For ~he amphipod ~est, samples were taken in solvent rinsed plastic syringes, and the solutions were pre-concentrated on Sep Pak

. cartridges (Waters Assoc., Millford, HA). The Sep paks were eluted with acetonitrile and the eluted sample injected. The samples for all other tests
were injected directly into the HPLC, generally on the same day they were taken. A few samples were run the following day because of equipment problems, but concentrations were calculated using standards stored inthe
same way. Manual injections were made by pumping the measured amount of sample or standard through aC-18 guard column which replaced the loop on the injector. The guard column was rinsed with deionized water before injecting and the system was rinsed between injections with acetonitrile followed by deionized water. The acute flow-through tests were sampled as follows: all chambers at 0, 48 and 96 hr, and half the tanks (one set of replicates) at 24 and 72 hr. The cladoceran acute test was sampled as follows: fresh solutions from preparation flask at Dand 24 hr, and old solutions from pooled replicates at 24 and 48 hr. The cladoceran chronic test was sampled as follows: fresh solutions on the days they were prepared (MWF) and old solutions from pooled replicates when they were replaced (MWF) and at termination of exposure. The rainbow trout chronic test was sampled twice per week inall test chambers. During the rainbow trout test, a change inthe injector rinsing procedure between samples resulted in the detection limit changing from 4.7 ~g/L before day 47 of the tesltto 10.1 ~g/L after day 47. When exposure chamber concentrations dropped below the detection limit one-half the detection limit was used as the solution concentration of nonylphenol. Mean exposure concentrations were calculated using this technique. For all tests, at least 10\ of the samples
were run l.nduplicate and at least 10\ were spiked tomeasure percent recovery of nonylphenol (Table 3). All nonylphenol concentrations used inthis report have been corrected for spike recovery.



### 9

~..Statisti.cal Analvsis of Data LC50 and ECSO calculations with their 95\ confidence limits were calculated by the trimmed Spearman-Karber method (Hamilton, et ale 1977) (Table 5). Chronic and plant test exposure NOECs and LOECs were determined by 

### aone-wayanalysisofvariance (ANOVA) test. Concentrations causing significant differences from controls ingrowth or reproduction were identified by Dunnett's test (1-sided) at a=O.05.



### RESULTS Acute Tests
~AdUlt ann::ids (meanweight 0.005 g) were exposed to five concentrations

# ~ of nonylphenol, plus control, induplicate in flow-through exposures. Mean measured nonylphenol concentrations ranged from 115 to 554 ~g/L. Effects

(inactivity) were observed inthe highest exposure concentration (554 ~g/L) at 24 hr, but deaths didnot begin to occur until 48 hr at this concentration (Table 4).' Mortalities at 96 hrwere 90\ at the highest concentration (554 ~g/L) and 55% at the second highest concentration (351 ~g/L). No mortality occurred in the remaining three exposures or control. The 96-hr LC50 estimate (95% confidence limits) is 342 (296-396) ~g/L (Table 5). The 96-hr ECSO is 268 ~g/L but confidence limits could not be computed because none of the exposure concentrations resulted inpartial effects. Adult snails (meanweight 476 Z 218 mg) were exposed to five concentrations of nonylphenol, plus control, induplicate in flow-through exposures. Mean measured nonylphenol concentrations ranged from 155 to 2,230 ~q/L. Effects (unable to retract into shell with gentle prodding) were observed prior to death inthe highest three concentrations (S30, 1,130 and 2,230 ~g/L). At 48 hr (Table 6), 100\ mortality had occurred in the two highest concentrations (1,130 and 2,230 ~g/L). Nomortality occurred in the remaining three exposures or control. The 96-hr LCSO estimate for Physella virgata is 774 ~g/L andthe 96-hr ECSO estimate is 378 ~g/L (Table 5). The 

### ~confidence limitswerenotreliablebecausenoneofthetest
10

### --------

concentrations of nonylphenol resulted inpartial mortality or effects. Dragonfly nymphs (meanweight 285 ~ 126 mg) were exposed to five concen~rations of nonylphenol, plus control, induplicate in flow-through exposures. Mean measured nonylphenol concentrations ranged from 46.0 to 768 ~g/L. Effects (loss of equilibrium and posterior of abdomen flexed over back)
were observed inthe highest four concentrations (103, 177, 434 and 768 ~g/L) within 24 hr of the initial exposure (Table 7). Mortalities were observed at 72 hr inthe highest exposure with 40% dead at 96 hr. The percent affected, including mortalities, were 70\ at the highest (768 ~g/L), 25\ at the second highest (434 ~g/L), 20% at the third highest (177 ~g/L), 10% at the fourth highest (103 ~g/L) and 15\ at the lowest concentration at 96 hr. Nomortality
or effects were observed in the control. The 96-hr LC50 for ophiogomphus sp. could not be estimated because fewer than 50\ of the organisms died at any exposure concentration during the test, but the ECSO estimate (95\ confidence limits) is 596 (497-714) ~g/L (Table S). Amphipod juveniles (approximately 2mm intotal length) were exposed to £ive concentrations of nonylphenol, plus control, induplicate in flow-through exposures. Mean measured nonylphenol concentrations ranged from 6.49 to 149 ~g/L. Mortalities occurred in the two highest concentrations (52.S and 149 ~g/L) within 24 hr of initial exposure (Table 8). No mortality occurred in the control. The 96-hr LC50 estimate (95\ confidence limits) forHyalella
az~eca is 20.7 (16.8-25.6) ~g/L (Table 5). Some organisms showed lack of mobility at nonylphenol concentrations ~l2.4 ~g/L during the test. However, at 96 hr there were no surviving organisms exhibiting these effects and the 96-hr ECSO estimate is the same as the LCSO estimate. Fathead minnows (mean standard length, 17.1 

### ~ 3.8 mm, mean weight, 81.6

~ 31.0 mg) were exposed to five concentrations of nonylphenol, plus control, in duplicate in flow-through exposures. Mean measured nonylphenol concentrations ranged from 61.8 to 264 ~g/L. Deaths began occurring within 24 hr of the test start inthe highest (264 ~g/L) exposure and at 48 hr in the second and third highest (109 and 160 ~g/L) exposures (Table 9). At 96 hr, 

### 11a 

### therewas 100%mortalityinthehighestconcentration (264 

### pg/L), 85%inthe second highest concentration (160pg/L) and 30% in the third highest concentration (109 pg/L). Nomortality occurred inthe lowest two exposures or control. The 96-hr LC50 estimate (95\ confidence limits) for Pimephales promelas is 128 (117-141) pg/L (Table 5). Some fish at exposure concentrations ~109 pg/L showed sublethal effects of loss of equilibrium and poor fright and schooling responses. The calculated 96-hr EC50 estimate is 96.0 pg/L. Confidence limits could not be calculated. Bluegill juveniles (mean standard length, 23.0 

### ~ 4.0 mm; mean weight, 294 

### ~ 209 mg) were exposed to five concentrations of nonylphenol, plus control, induplicate in flow-through exposures. Hean measured nonylphenol concentrations ranged from 35.3 to 384 pg/L. Deaths began occurring within 24 hr of the test start inthe highest (384 pg/L) concentration and within 48 hr in the second highest (192pg/L) concentration (Table 10). At 96 hr, there
was 100% mortality inthe highest concentration (384pg/L) and 45% inthe second highest concentration (192pg/L). Nomortality occurred inthe remaining three exposures or control. The 96-hr LCSO estimate (95\ confidence lLmits) for Lepomis macrochirus is 209 (184-238) pg/L (Table 5). Some fish at nonylphenol concentrations ~192 pg/L showed 10SB of equilibrium and loss of schooling ability prior to death. The computed 96-hr EC50 estimate (95% confidencE! limits) is 203 (178-231) pg/L. Rainbow trout fingerlings (mean standard length, 27.2 

### Z 2.5 mm; mean weight, 241

### ~ 64.9 mg) were exposed to five concentrations of nonylphenol, 

### pluscontrol, induplicateinflow-throughexposures. Measurednonylphenol concentrations ranged from 16.0 to 344 pg/L. Deaths began occurring within 24 hr of initiation of the test with 100\ mortality inthe highest (344pg/L) exposure at 96 hr (Table 11). One death occurred, and one fish was affected in the third highest (8S.1pg/L) exposure on the last day. All of the fish in the second highest (152pg/L) exposure began to show effects at 48 hr after test initiation. Nomortality or effect was observed in any of the other exposures or control. The 96-hr LC50 estimate (95% confidence limits) for


### 12Oncorhynchus mykiss is 221 (208-236) ~gfL (Table 5). Sublethal effects recorded were loss of equilibrium or the loss of ability to react to a fright response. The 96-hr EC50 estimate (95% confidence limits) is 109 (100-118) ~g/L. Duckweed (2 leaf fronds per plant) were exposed to five concentrations of nonylphenol, plus control, induplicate in flow-through exposures. Hean measured nonylphenol concentrations ranged from 109 to 2,080 ~gfL. Frond production (new fronds) at 96hrwas significantly (a=0.05) reduced (62%of the control) only inthe highest concentration (2,080 pgfL). The remaining exposure concentrations had frond production that was 76% ormore of the control production (Table 12). The 96-hr highest observed no effect concentration (NOEC) is 901 pgfL and the lowest observed effect concentration (LOEC) is 2,080 ~gfL (Table 5). The geometric mean of these values is 1,369 

### ~g/L. Cladocerans «24-hr old neonates) were exposed to five concentrations of nonylphenol, plus control inquadruplicate. Mean measured nonylphenol concentrations (mean of 0 and 24 hr new solutions) ranged from'22.1 to 533 ~g/L. During the 24 hr between renewal of test solutions, nonylphenol concentrations declined an average of 24.8 % 5.35% with no relationship in percentago decrease with exposure concentration. Deaths began occurring within 24 hr of the initial exposure with 100%mortality in the two highest

(248 and 533 pgfL) exposures at this time (Table 13). After 48 hr of exposure to nonylphenol there was 65% mortality in the third highest (126pgfL) exposure. No deaths occurred inthe remaining exposures or control. The 48- hr ECSO estimate (95% confidence limits) forDaphnia magna is 84.8 (76.0-94.6) pgfL (Table 5). Green algae were exposed to seven concentrations of nonylphenol, plus control intriplicate static exposures. Mean measured nonylphenol concentrations ranged from 26.6 to 2720 pgjL. Concentrations were measured in surrogate test chambers (containing no algae) measured at 0, 48 and 96 hr. During th(~96 hr test, nonylphenol concentrations declined an average of 50.4\


### 13for the concentrations of 66.7 through 1,480 ~g/L. The lowest test concentration (26.6 ~g/L) and the highest (2,720 ~g/L) decreased by 86.5 and

. 74.2%, renpectively. Concentrations of nonylphenol at 96 hr appeared disproportionately low inboth cases. Cell production (bydryweight) was 30\ and 0\ of control inthe two highest (1,480 and 2,720 ~g/L) nonylphenol exposures, respectively. At these two exposure concentrations, cell production was significantly less than control at a=0.05 (I-sided) by Dunnett's test (Table 14). The cell production was at least 88\ of control dry weight and not significantly different from control inany of the remaining exposures. The NOEC 

### forSelenastrumcapricornutumexposedto nonylphenol is 694 ~g/L and the LOEC is 1,480 ~g/L (Table 5) and the geometric 

### meanofNOEC 

### andtheLOEC

### is1,013~g/L.

Chronic Tests Three attempts were made to conduct a chronic 21-day test solution renewal exposure of nonylphenol with the cladoceran, Daphnia magna before a test in full compliance with ASTM (199Id) testing guidelines was accomplished. All three tests are described because the results from each agree well with one another. Daphnia magna «24-hr old neonates) were exposed inTest Number 1 to five concentrations of nonylphenol, plus control. Measured nonylphenol concentrations ranged from 31.0 to 245 ~g/L. Between solution renewals, nonylphenc,l concentration declined an average of 48.0 ~ 9.50%. Ten replicates with one neonate per replicate were used for each exposure and control. Deaths began occurring within 48 hr of the initial exposure with 100% mortality in the highest (245~g/L) at test termination (Table 15). Percentage survival of adults and number of young produced at the termination of exposure was not significantly (a=0.05) different from the control at any of the remaining concentrations (Table 15). Reproduction began on day 11-12 at all concentrations and control, except inthe highest (245 ~g/L) which had 

### no livingadultsbydayfive.

.The NOEC 

### for

## D.magnaexposedtononylphenolis
1499.5 ~g/L and the LOECbased upon survival of adults is 245 ~g/L for Test Number 1 and the geometric mean (chronic value) of the NOECand the LOEC 

# . is 156.1 ~g/L. 

### Dapl!nia magna «24-hr-old neonates) were exposed in Test N~er 2to fiveconclE~ntrations of nonylphenol, 

### pluscontrol.Measurednonylphenol concentra1:ionsrangedfrom11.2to 165~g/L. Nonylphenolconce~trations decreasedanaverageof55.3 

### : 17.0\betweenrenewaloftestsolutions.Ten replicate~1withone neonate perreplicatewereusedfor eachexposureand control. Deaths began occurring within 48 hr of the initial exposure with 70\ mortality in the highest (165 ~g/L), 50\ in the second highest (77.3 ~g/L), 30% in the third and fourth highest (17.7 and 38.8 pg/L), 50\ in the lowest (11.2 ~g/L) and 40\ inthe controls at termination of exposure (Table 16). The high mortality inthe controls and lower exposures invalidated the test. Reproduction began by day 8 at all concentrations and control, except inthe highest (165 ~g/L) exposure. The number of young produced at the termination of exposul~ewas significantly (a=0.05) different from the control only in the 

### highest (165~g/L) exposure. The NOEC

### forD.magnaexposedtononylphenolis 77.3pg/L and the LOECbased 

### uponreproductionis165~g/Landthegeometric mean (chronicvalue) of the NOECand the 

### LOECis112.9~g/L. Dapl!niamagna «24-hr-old neonates) were exposed inTest Number 3 for 21 days to five concentrations of nonylphenol, plus control. Ten replicates with one organism per replicate were used for each exposure and control. Measured nonylphenol concentrations (mean of new test solutions) ranged from 44.3 to 500 ~g/L. Nonylphenol concentrations declined an average of 57.4 

### : 5.79\ between renewal of test solutions. Deaths began occurring inthe two highest (215 and 500 ~g/L) within 48 hr of initial exposure with 40\ and 100\ mortality, respectively, at test termination. Percentage survival of adults at the termination of exposure was not significantly different from the control il1the remaining exposures (Table 17). Reproduction began ondays 7-9 at all cOllcentrations and control, except in the highest (500 ~g/L) exposure which had no living adults by day 2. The mean 

### lengthof survivorswas 

### 15significantly different from controls in the second highest (215 ~g/L) exposure, but not in the three remaining exposures (Table 17). The NOEC for D. magna exposed to nonylphenol is 116 ~g/L, and the LOEC based upon reproduction is 215 ~g/L (Table 5). The geometric mean of the NOEC and the LOEC (chroni~ va~ue) ~orthis test is 157.9 ~g/L. The results from the three tests agree well, but only Test Number 3 is used to calculate a chronic value. Rainbow trout (freshly fertilized ova), were exposed to five concentrations of nonylphenol, plus control, induplicate. Mean measured nonylphenol concentrations ranged from 6.0 to 114 ~g/L for the flow-through test. Exposures lasted for 91 days with hatching occurring at 34 ~ 5 days for all nonylphenol concentrations. Percent hatch was not affected by any of the tested nonylphenol concentrations (Table 18). Deaths began occurring one day post-hatch with 100\ mortality inthe highest (114 ~g/L) and 98.5\ mortality in the second highest (53.0 ~g/L) exposure by 3 days post-hatch. There was 100\ mortality in the second highest (53.0 ~g/L) exposure by 13 days posthatch when the fry in the remaining exposures were transferred from the incubation cups. Deaths began occurring by 8days post-hatch inthe third highest (23.1 ~g/L) exposure (5days after transfer). There was 66\mortality in the third highest (23.1 ~g/L) exposure by the end of the test. The mortalities inthe remaining two exposures were not significantly different from the control. Mean standard length andwet and dryweights at the termination of the test were all significantly (a=O.OS) different from control at concentrations of 10.3 and 23.1 ~g/L (Table 18). No significant effects
were observed at the 6.0 ~g/L concentration. The NOEC for oncorhynchus mykiss 

### exposedtononylphenolis6.0~g/L, andtheLOEC

### basedupongrowthis10.3 ~g/L (Table5). ThegeometricmeanoftheLOECandthe NOEC (chronic value) 

### forthistest is7.861~g/L.

16

0-REFERENCES

ASTM. 1991a. Standard guide for conducting acute toxicity tests with fishes, macroinvertebrates, and amphibians. E 729-88a. American Society for Testing and Materials, 1916 Race Street, Philadelphia, PA. Annual Book of Standards. Vol. 11.04 pp. 378-397.

ASTM. 1991b. Standard guide for cond~cting static 96-h toxicity tests with microalgae. E 1218-90. American society for Testing and Materials, 1916 Race Street, Philadelphia, PA. Annual Book of Standards. Vol. 

### 11.04. pp. 845-856.

ASTM. 1991c. Standard guide for conducting static toxicity tests with Lemna gibba G3. E 1415-91. American society for Testing and Materials., 1916 Race Street, Philadelphia, PA. Annual Book of Standards. Vol. 11.04. pp. 1137-1146.



### ASTM. 1991d. Standardguideforconductingrenewallife-cycletoxicitytests withDaphniamagna.E 1193-87. AmericanSocietyforTestingand Materials, 1916RaceStreet,Philadelphia,PA. AnnualBookof Standards. Vol. 11.04. pp. 845-856.
Benoit, D.A.,V.R. MattsonandD.L. Olson. 1982. Acontinuous-flowminidilutersystemfortoxicitytesting. WaterRes. 16:457-464.
Bhatt, B.D., J.V. Prasod,G. KalapanaandS.Ali. 1992. Separationand characterizationofisomersofp-nonylphenolsbycapillaryGC/GC-MS/GCFTIRTechniques. J. Chromatogr.Sci. 30:203-210.
Naylor, C.G. 1993. TexacoChemicalCo.,Austin,TX. (MemorandumtoLarry Brooke, University of Wisconsin-Superior, Superior, WI, January, 29.) 

### 17stephan, C.E., D.l. Hount, D.J. Hansen, J.H. Gentile, G.A. Chapman, and W.A. Brungs. 1985. Guidelines forderiving numerical national water quality

. criteria for the protection of aquatic organisms and their uses. U.s. Environmental Protection Agency, Office of Research and Development. PB 85-2270. National Technical Information Service, 5285 Port Royal, 

### Springfield,VA22161. 98pp.

18

0--TABLE 1. Water Characteristics of Nonylphenol Exposures toVarious Freshwater Organisms. 

## Mean.:t Standard Deviation, Range (inParentheses) andNumber of Observations (n). Dissolved Total Alkalinity Specific Organism & Temperature Oxygen Hardness (mg;Las pH Conductivity Test Typel! (C) (mg/L) (mg/Las (

### caco)) (llmhos/cm) 

### CaCo,) Rainbowtrout 107:t0.7 

### 9.4:t1.1 54.0:t6.4 41.0.:t3.3 

### 6.97.:t0.39 120.:t12 FT,Chronic (8.9-14.4) (7.7-11.7) 

### (39.0-66.3) (32.0-52.0) (6.34-7.84) (97-13B) n=980 n=102 n-42 n=42 n=98 n=BO Rainbowtrout 11.9+0.4 10.0;t0.4 56.6;t2.3 44.0.:t0.0 6.72;t0.16 95;t5 FT, Acute (11.3=12.6) (9.3-10.4) (54.6-58.5) (44.0-44.0) (6.53-6.99) (90-100) 

### n=60 n=12 n=4 n-4 n=12 n=8 Fatheadminnow 22.4;t0.4 7.7.:t0.5 57.3;t4.7 47.5.:t2.9 7.23.:t0.31 149.:t1 FT, Acute 

### (21.3-23.1) (6.6-9.2) (53.6-63.4) (45.0-50.0) (6.77-7.59) (149-150) n=52 n=12 n=4 n=4 n=12 n=4 Annelid 23.7;t1.2 7.8.:t0.4 52.7;t2.3 44.0;1:0.0 6.75:t0.l0 146:t2 FT, Acute 

### (22.2-25.9) (7.2-8.5) (50.7-54.6) (44.0-44.0) (6.63-6.90) (145-148) n=60 n=12 n..4 n=4 n=12 n=4 Bluegill 22.7;1:0.4 7.9.:t0.3 51.7.:t2.0 45.5:t1.0 7.61.:t0.21 144.:t2 FT, Acute (21.5-23.8) 

## (7.5-8.2) (50.7-54.6) 

### (44.0-46.0) (7.26-7.79) (142-145) n=60 n=12 n..4 n=4 n=12 n=4 Amphipod 24.6;1:0.5 8.2;1:0.4 51.5.:t5.0 45.3.:t1.9 7.80.:t0.11 136.:t3 FT, Acute (23.7-25.6) (7.6-8.B) 

### (46.0-60.0) (42.4-48.0) (7.69-8.01) (130-140) n=60 n=11 n=12 n=12 n=12 n-12 Snail 23.7+0.5 8.0;t0.2 54.5;1:3.7 47.7.:t3.1 7.89.:t0.10 132.:t7 FT, Acute (22.9=24.5) 

### (7.8-8.2) (50.0-60.0) (42.0-52.0) (7.73-a. 02) (125-140) n=40 n=12 n=8 n=8 n=12 

## n-a Dragonfly 23.3j:1.1 8.4j:0.4 51.7j:2.0 43.0j:3.8 8.06j:0.21 ND3/ FT, Acute (21. 5-25.0) (7.7-8.9) (50.7-54.6) (40.0-48.0) (7.82-8.52) n=60 n=12 n..4 n=4 n"12 Duckweed 22.8j:0.3 7.6j:0.6 175;1:28.7 105j:22.6 S.2Sj:0.13 ND FT, Acute (22.8-24.2) (6.5-8.5) (108.0-210.0) (68.0-132.0) (8.01-a.67) n=60 n=60 n=17 

### n=17 n=60TABLE 1. (Cont~)

II V 3' FT=flow-through; R=renewal; S-static. Determined for algae media. Not Determined. Cladoceran 20.S;t0.5 .8.7;t0.S 167;t9.2 120;t9.1 8. 46;t0. 11 536;t21 R, Chronic (19.7:t21.8) (7.5-9.6) (156.0-18!:l.0) (101.0-144.0) {S.H-a.5s} (490-600; n=90 n=92 n"16 n=16 n=95 n=92 Cladoceran 22.0;t0.5 7.6;t0.6 184;tl0.3 104;t0.0 7.87;t0.13 627;t70 R, Acute (21.0-23.0)

. (6.3-8.2) (175.6-195.1) (104.0-104.0) (7.66-8.04 ) (525-700) n=20 n=10 n=4 n=4 n=7 

### n-l0 Green Algae 25.7;t0.2 

### ND 16.0v 54.0v 7.49;t0.12 ND 5, Acute (25.2-26.0) (7.30-7.70) n=32 n-40

### TABLE 2. summary ofAnalytical Conditions Used forAnalysis of Nonylphenol Toxicity Tests

FlowRate 

### Pre- Sample Detector Test Organism Test Typell Mobile Phase (mL/min) Concentration Size AUFS2I Wavelength (nm) Annelid 

### A/FT 40\ CH,CN, 40\ CH)CN 

### 1.5 Yes 800pg/L 1.000 275 20\0.1.H20,5\THF Snail A/FT 40\ CH,CN, 40\ CH,OH, 

### 1.5 No 600pL 1.000 275 15\0.1.H20,5\THF Dragonfly A/FT 40\ CH]CN, 40\ CH,OH, 

### 2.5 No 600pL 1.000 275 15\D.I.H20,5\THF Amphipod A/FT 40\CH]CN,40\CH)OH, 1.5 Yes 30mL 1.000 275 20\ D.I. H2O Fathead Minnow 

### A/FT 40\CH]CN, 40\ CH,OH, 

### 1.5 No 600pL 1.000

. 

### 275 15\0.1.H20,5\THFY Bluegill A/FT 40\ CH]CN, 40\ CH]OH, 

### 2.5 No 600pL 1.000 275 15\0.1.H20,5\THF RainbowTrout A/FT 95\ CH]CNor CH]OH, 

### 1.0 No 600pL 1.000 275 5\D.I. HP 

### RainbowTrout C/FT 40\CH]CN,40\CH]OH, 1.5 Yes 10mL 1.000 275 15\D.I.H20,5\THF Duckweed A/FT 40\CH)CN,40\CHpH, 1.3 No 300pL 0.500 224 20\ D.Ie H2O 

### Cladoceran CjR 80\CH]CN, 20\ D.I. H2O 

### 1.0 No 300pL 0.250 224 GreenAlgae A/S 80\CH)CN, 20\ 0.1. Hz<> 

### 1.0 No 300pL 0.250 224 II A=acute; C=chronic; FT=flow-through, S-static; R=renewal. 21 AUFS

## =Absorbance Units Full Scale. Y THF=Tetrahydrofuran.11 '1J A=Acute test; C=Chronic test; R=Renewal S=Static; FT=Flow-through Calculated by the u.s. EPA recommended method published inFoderal Regiscer Vol. 49, No. 209, October 26, 1984. Two D.L. calculated due to achange in instrument sensitivity midway through 

### thetest.

. 

# "

TABLE 4. 

### Dead/AffectedAnnelids, Lumbriculus variegacus, Exposed to Five Concentrations of Nonylphenol Over Time. Twenty Organismsper ExposureConcentration.

Mean Exposure Concentrations : S.D. (J.lg/L) 

### Time(h)


### o 24 48 72 96 Control <69.2 

## o o o o o <69.2 

# o o a o o 115:32 o o o o o 204:1:28 

# a o o o o 351:91 o o o 0/20 11/9 554:1:91 

### o 0/20 1/19 2/18 18/2

22

L. 

### TABLE3. Agreement of Duplicates, Spike Recoveries and Detection Limits for the Nonylphenol Exposures of Selected Freshwater Organisms

Test Mean Standard Deviation Detection Organism Type" %Agreement %Recovery Limit (J.lg/L)'1J Fathead minnow 

### A/FT 87.5 12.4 93.7 15.9 37.5 Snail A/FT 93.8 ;t4.1 96.1 14.7 88.0 Cladoceran 

### A/R 94.1 3.0 90.1 ;t7.3 7.3 Cladoceran C/R 93.7 1.9 76.0 j:12.1 6.8 Bluegill A/FT 89.4 7.2 80.1 j:26.9 29.7 Dragonfly 

### A/FT 90. 4 j: 7 ..8 

### 75.7;t23.0 29.7 Duckweed A/FT 94.8 j: 2.3 

### 98.2 ;t 

### 18.1 88.0 Rainbow Trout 

### A/FT 89.0 ;t7.8 88.7 ;t16.4 17.2 Rainbow Trout C/FT 91.5 7.4 91.5 j:25.3 4.7 10.1" Annelid A/FT 93.6 j:9.1 74.0 ;t12.9 69.2 Amphipod A/FT 

### 83.1 .t

### 12.5 100.7 j: 

### 13.3 2.3 Green Algae A/S 

### 94.3 .t

## 5.0 75.3 6.3 

### 6.811 S=static; FT=Flow-througb; R=Renewal; All tests with measured concentrations of nonylphenol. 95% confidence limits are not reliable. '1J Lowest Observed Effect Concentration based upon number of fronds produced. 

### Geometricmeanof 

### NOECandLOEC. Lowest Observed Effect Concentration based upon biomass. Lowest Observed Effect Concentration based upon mean length of surviving 

### adults. Lowest Observed Effect Concentration based upon total length, andwet anddry weights. 11

23



### t- , TABLE S. Effect Concentrations ofAcute andChronic Exposures ofNonylphenol toVarious Freshwater Organisms.

'tTestType '-2\ Organism Test Effect 

# 05, Duration Type Concentration (g/L) ACUTE 

### !rESTS Annilid, FT (96h) LCSO 

## 342,/ (296-396) 

### Lumbriculusvariegatus ECSO 268/' 

### ( '1J 

### ) Snail, 

### FT (96h) LCSO 774 / ( '1J 

### ) Physellavirgata ECSO 378r ( 31 ) Dragonfly, FT (96h) EC50 596 ./ (497-714) Ophiogomphusap. Amphipod, FT (96h) LCSO 20.7

## : (16.8-25.6) Hyalellaazteca ECSO 20.7 (16.8-25.6) Fatheadminnow, FT (96h) LC50 

### 128

'" (116-141) Pimephales promelas EC50 96.0/ ( '1J) Bluegill, FT (96h) Le50 

### 209,.- (184-238) Lepomis macrochirus EC50 203/ (178-231)

,.- Rainbow trout, 

### FT (96b) LC50 221 (208-236) Oncorhynchus mykiss ECSO 

### 109

..- (lOO-118) Cladoceran, R (48h) EC50 84.8/ (76.0-94.6) Daphnia magna Duckweed, FT (96h) NOEC 901 r 136941 Lemna 

### minor LOEe" 2080-/ Green algae, S (96h) NOEC 

### 694

........ 101341 Selenasrum capricornutum LOEeS' 1480 CHRONIC !rESTS Cladoceran, 

### R (21d) HOEC 116/ 157.941 Daphnia magna LOEe" 215/ Rainbow trout, FT (90d) BOEC 

### 6.0 7.86141 Oncorhynchus mykiss LOEC7t 10.3TABLE 6. 

### Dead/Affected Snails, Physella virgata, Exposed to Five ConcentrationsofNonylphenolOverTime. TenOrganismsperExposure Concentration.

TABLE 8. 

### Dead/Affected Amphipods, Hyalella azteca, Exposed to Five Concentrations of Nonylphenol Over Time. Twenty Organisms per ExposureConcentration.

24



### .- MeanExposureConcentrations%S.D. (lJg/L) Time(h) Control <88 155%118 269:t91 530:tl43 1,130:!:246 

### 2,230:390 0 0 0 0 0 0 0 48 0 0 0 0 10 10

-- 

### 96 0 0 0 0/10 10 10

TABLE 7. Dead/Affected Dragonfly Nymphs, Ophiogomphus sp., Exposed to Five Concentrations of Nonylphenol Over Time. Twenty Organisms per Exposure Concentration.

Mean Exposure Concentrations : S.D. (ua/L) (\ \ Time(h) Control G68J239 \ 

### <29.7 46.0:!:36.6 103:!:88.1 177:1:86.0 434:!:l54 \ 

### \ 0 0 0 0 0 0 0 \ \ 24 0 0/1 0/1 0/7 0/11 I 0 J 

### 48 0 0 0/1 0/0 0/2 0/2 72 0 1/1 0/3 0/3 0/5 5/5 96 0 2/2 0/2 0/4 0/5 8/6 i

Mean Exposure Concentrations :I: S.D. (lJg/L) Time(h) Control <2.30 6.49:3.12 12.4:1:5.96 21.8:t7 .84 S2.5:!:lS.1 149:!:48.3 

### 0 0 0 0 0 0 0 24 0 0 0/1 0/1 7/1 9/7 48 0 0 1/0 1/0 16/2 20/0 72 0 0 2/1 4/1 18/2 20 96 0 1/0 3/0 11/0 20/0 20TABLE 9. Dead/Affected 

### FatheadMinnows, 

### Pimephales promelas,ExposedtoFive Concentrations of Nonylphenol Over Time. Twenty Organisms per ExposureConcentration.

TABLE 10. Dead/Affected Bluegill, Lepcmis macrcchirus, Exposed to Five COncentrations of Nonylphenol Over Time. Twenty Organisms per ExposureConcentration.

TABLE 

### 11. Dead/AffectedRainbowTrout, Oncorhynchusmykiss, ExposedtoPive Concentrations of Nonylphenol Over Time. Twenty Organisms per Exposure Concentration.



### 2S MeanExposureconcentrations 

### S.D. (J.lg/L) Time(h) Control <37.5 61.8:12.2 84.519.l 109:23.3 160:22.2 264:33.6 0 0 0 0 0 0 0 24 0 0 0 0 0 13/7 48 0 0 0 2/18 8/12 8/12 72 0 0 0 6/14 15/5 20 96 0 0 0 6/14 17/3 20

Mean Exposure Concentrations :tS.D. (1l9/L) Time (h) 

### Control <29.7 35.3:19.7 71.4:UO.7 120:U3.6 19225.2 384:t49.4 

### 0 0 0 0 0 0 0 24 0 0 0 0 0 2/1 48 0 0 0 0 1/0 16/4 72 0 0 0 0 5/0 20 96 0 0 0 0 9/1 20

Mean Exposure Concentrations : S.D. (J.lq/L) Time(h) 

### Control <17.2 16.0:9.7 45.0:28.8 88.1:t23.7 152:t2S.1 344:t46.0 

### 0 0 0 0 0 0 0 24 0 0 0 0 0 5/15 48 0 0 0 0 1/0 15/5 72 0 0 0 0 0/20 19/0 96 0 0 0 111 0/20 20TABLE 

### 12. Frond Production for the Duckweed, Lemna minor, Exposed to Five Concentrations of Nonylphenol Over Time. Twenty-two Frond Plants Were Introduced per Exposure Concentration.

TABLE 13. Affected Cladocerans, Daphnia magna, Exposed toFive Concentrations of Nonylphenol OVer Time. Twenty Organisms per Exposure Concentration.



### 26 MeanExposureConcentrations%S.D. (1l9/L) Time(h) Control <88.0 <88.0 109%38.8 3S%80.8 90U112 2,080=188 0 40 40 40 40 40 40 24 44 46 46 45 44 40 48 5S 50 50 55 48 41 72 69 60 60 66 S3 47 96 77 72 68 74 68 63°

, Significantly less than the control plants at a=O.OS.



### MeanExposureConcentrationsII% S.D. (1l9/L) 

### Time(h) Control <7.3 22.1:t10.2 

### 48.5%7.07 126%5.66 248%0 533%0 0 0 0 0 0 0 0 24 0 0 0 3 20 20 48 0 0 0 18 20 20 11

### Meanofnewsolutionnonylphenolconcentrations.TABLE 14. 

### Mean Growth (Dry 

### Weight Biomass) of cultures of the Green 

### Algae, Selenastrumcapricornutum, after 96-Hour Exposures to Various Concentrations of Nonylphenol. Initial Chemistry was 2 x 105Cells per Milliliter in 100 Milliliter Test Solutions.

Significantly different from the control at aKO.OS.



### 27 

## Mean:tS.D. Mean :tS.D.

Exposure Dry Weight (mg) Concentration of Algal Cells in (JJg/L) Exposure Flasks



### n=3 n=3 Control

<6.8 8.8 :!:0.6 26.6 :t:19.6 8.3 :t0.4 66.7 :!:22.2 8.5 :t0.7

136 :t48.0 9.4 :t1.0 329 :t:84.8 8.0 :t:1.3 694 :t:247 7.7 :!:0.8 1,480 :t552 2.6 :t2.48 2,720 :t1,570 0.3 :t0.18TABLE 15. survival andReproduction ofthe Cladoceran, Daphnia magna, Exposed to Various Concentrations of Nonylphenol for 21 Days
- Test 1.



### Percent survivalof 

### adults Mean number of young/surviving adult after 21 

### days Mean nU111.berof 

### young/st,uting adult 36.8 58.8 54.4 52.0 31.0
Significantly different from control at a=0.05.

TABLE 

### 16. Survival andReproduction of the Cladoceran, Daphnia magna, Exposed toVarious Concentrations of Nonylphenol for 21 Days
- Test 2.



### 28 Mean Nonylphenol Concentrations % S.D. (lJg/L) Control

<18.7 <18.7 31.0%3.94 53.9:1:7.84 99.5:1:5.98 245%58.4 90 90 100 90 90 0

32.9 61.8 53.3 55.0 29.8 O.

Mean Nonylphenol Concentrations :I:S.D. (lJg/L) COntrol

<2.3 11.2:1:3.88 17.7:1:6.82 38.8%15.0 77.3:1:29.1 165:1:58.6 

### Percent 60 SO 70 70 SO 30 survivalof adults Meannumberof 94.2 106 80.1 87.8 94.0 28.3. young/surviving adultafter21 days Meannumberof 88'.2 81.4 77.3 79.9 67.0 10.O. young/starting adult Significantlydifferentfromcontrolata=0.05.TABLE 17. Survival andReproduction of the Cladoceran, Daphnia magna, Exposed to Various Concentrations of Nonylphenol for 21 Days
- Test 3.



### 29

.- Mean Measured Nonylphenol COncentrations % S.D. (pg/L) Control

<6.8 44.3:Ul.l 63.1:t:9.34 

### 116tl20 21S:U6.9 500:1:0 Percent 80 90 100 90 60 O. survivalof adults MeannW1\berof 111 113 103 118 87.5 O. young/surviving adultafter21 days Meannumberof 94.8 102 103 116 53.3" O. young/starting adult Mean length 3.94 3.93 3.84 3.84 3.62" 0" (mm)of surviving adults Significantlydifferentfromcontrolata-O.OS..-. nA~~lIaUL.~CYI 

### ADnorma~Development,SurvivalandGrowthofRainbow Trout (Oncorhynchus mykiss) Exposed toNonylphenol for 91Days PostFertilization.

Mean Nonylphenol Concentrations ~ S.D. (~g/L)

II Detection Limit

- 4.7 ~g/L for first half of test and 10.1 ~g/L for second half of test. Live fry/total eggs atday 40. At three days post-hatch. Percent survival of 30 iridividuals transferred from egg cups to exposure chambers at 13 days post-hatch. Significantly different from control at a = 0.05. Dry weight (g) of total survivors in chamber. 

### Sf

~

30

- - - - <D.L.II6.02.5 10.3:tS.4 

### 23.U5.8 53.0%12.7 114:t21.5 

### Mean rcent 83.5 89.0 88.5 92.0 92.5 88.5 hatch Heanpercent 3.5 2.0 1.0 0.5 95.5 100 abnormalordead at hatchJI 

### Meanpercent 93.3 93.3 90.0 33.351 0$1 051 

### survivalat51 dayspost-hatch Meanwetweight 0.734 0.716 0.516$1 0.375s1 0$1 

### 0"

(g)at 51days post-hatch Meandryweight 0.135 0.133 0.095" 0.066$1 051 OS1 

### (g)at 51days post-hatch Meanstandard 38.5 38.0 33.151 29.851 

### 0" 0" 

### length(Dim) 

### at51 dayspost-hatch Mean 1.89 1.87 1.28 0.33 0 0 biomass/chamber