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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

March 24'~

~o ~. r.
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Although the information described in this article has been funded wholly or

in part by the Environmental Protection Agency under contract 68-CI-0034

entitled "Technical support services for water quality criteria and related

activities," it does not necessarily reflect the views of the Agency an no official endorsement should be inferred.

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The Environmental Health Laboratory at the University of WisconsinSuperior (UW-S) conducted tests to complete the data set necessary to derive

national 1~ater quality criteria for nonylphenol. The tests were the eight

acute tests specified in the "Guidelines for deriving numerical national water

quality criteria for the protection of aquatic organisms and their uses"

(Stephan f!t ale 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 if both fresh and saltwater

criteria ",re to 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 a duckweed, 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 from ASTM 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

2
the 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 "in the 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 in the St. Louis River,

Douglas ~ounty, WI. They were cultured in battery 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 50 mL of inoculum consisting of

miscellaneous green algae from the previous culture water were added to each

jar. An udditional 20 mL of YTC was fed on the second and fourth day after

renewal. Organisms of about 2 mm total length (juveniles) were selected for

the test.

Fathead minnow embryos from brood cultures maintained at UW-S were

hatched and reared 25 to 35 days in flowing lab water. The fry were 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 a glass tube, counted and placed in random order into

3
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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 fry were 4S

days post-hatch.

Bluegill juveniles (~l yr-old) were acquired from Kloubek Fish Hatchery,

Amana, IA. They were acclimated in laboratory water for seven days prior to

test initiation and fed trout chow.

Cladocerans were acquired from ERL-D and cultured at UW-S in hard (160-

180 mg/L as CaC03) reconstituted water (ASTM 1991a) which was renewed three

times weekly (MWF). Cladocerans were fed YTC and a green algae (So

capricornu&um) on a MWF 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 a nutrient media (ASTM 1991b)

aseptically prepared. Nutrient media and inoculum were placed in a 4.0 L

Erlenmeyer flask, aerated, and stirred by magnetic 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 a hemacytometer for preparation of the desired cell

density to initiate the toxicity test.

Duckweed was obtained from the ERL-D culture. It was 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 by ASTM. These were found to have a better

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

4
sulfite 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

concentrations and a control, all in duplicate. Test chambers for all flow- .

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 (S x 15 cm) capped with neoprene stoppers and placed in the test

chambers. Amphipods were placed in 250 mL beakers with two 1.6 cm diameter

holes cov(lred with 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 fed during the exposure, and mortalities observed were

recorded daily.

Cladocerans were acut~ly exposed in 100 mL glass beakers containing 80

mL of test solution. . Four replicates were prepared for the control and each

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

5
control were used. Each replicate contained 100 freshly fertilized embryos.

The embryos were placed in oscillating incubation chambers consisting of 100 ,

mL beakers with the bottoms removed and replaced with nylon mesh (two per

replicate with 50 embryos in each). The incubation chambers were slowly raised

and lowered to facilitate movement of test solution around the embryos. The

organisms were kept in the oscillating incubation chambers without direct

lighting until swim-up of the fry occurred (approximately 45 days). Fifteen

fry were randomly chosen from the two oscillating incubation chambers and

released into each test chamber. At this time, photoperiod (16L:8D) was

initiated and the fry were fed. Feeding consisted of a ration of trout chow

three times a day. 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 hr prior to starting the test. A

single cladoceran «24-hr-old) was placed in each 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 a mixture of YTC and green

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 a mini-diluter that was modified to

receive toxicant and nutrient solution. The toxicant had a O.S dilution

factor but the nutrient solution was delivered equally to all test chambers.

The nutrients were essentially the same as in the 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

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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!lity or 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/mL at

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]c were filtered through a rinsed, 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 a desiccator and reweighed. Weight

corrections were made for flocculent present in the nutrient solution.

Temperature, total alkalinity, total hardness (EDTA) and pH were

determined in the test solutions (Table 1) for all tests. In most 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 in the control, low, medium and high exposure

concentrations, starting at test initiation for acute tests, and at least

twice a week in chronic exposures. Total hardness, total alkalinity, pH and

conductivity were determined at least once during an acute exposure and weekly

for the chronic exposures in control, low, medium and high exposure

concentrations.

All nonylphenol sto~k solutions were generated with a liquid/liquid

equilibrator (no organic solvents were used). Nonylphenol with a density less

than water was added to a container of test water (or nutrient 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

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flask and fresh water .was added to replace the solution removed by means 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 .two Waters H-45 pumps, ~waters

automated gradient controller, a Waters M-490 UV detector (Waters Associates,

Millford, MA), a Micromeritics Model 725 autoinjector (Micromeritics.

Instrument Corp., Norcross, GA) or a Valco C6W manual injection valve (Valco

Instrument Company Inc., Houston, TX) and.a Spectra-Physics SP4270 integrator

(Spectra-Physics, San Jose CA). A LiChrocart 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 [CAS No. 25l54-52-3} was obtained from Aldrich Chemical

Company (liilwaukee, WI) as a technical 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

stock solution was obtained by dissolving the chemical in acetonitrile (UV

Grade, Am"~rican Burdick & Jackson, Muskegon, HI). Standards were prepared

from this stock by dilution first in acetonitrile (UV Grade) and then in

Hilli-Q (~Iillipore Corp., 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 by glass pipet from the center of the test chambers

(or the center of the beakers within the chambers) at mid-depth and placed in

autoinjector vials or solvent rinsed, screw cap tubes for the manually

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injec~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 in the

same way. Manual injections were made by pumping the measured amount of

sample or standard through a C-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 D and 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 in all test chambers. During

the rainbow trout test, a change in the injector rinsing procedure between

samples resulted in the detection limit changing from 4.7 ~g/L before day 47

of the teslt to 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 to measure percent recovery

of nonylphenol (Table 3). All nonylphenol concentrations used in this report

have been corrected for spike recovery.

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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

a one-way analysis of variance (ANOVA) test. Concentrations causing

significant differences from controls in growth or reproduction were

identified by Dunnett's test (1-sided) at a=O.05.

RESULTS

Acute Tests

of nonylphenol,

~AdUlt ann::ids

plus control,

(mean weight

in duplicate

0.005 g) were

in flow-through

exposed to five

exposures.

concentrations

Mean

~

measured nonylphenol concentrations ranged from 115 to 554 ~g/L. Effects

(inactivity) were observed in the highest exposure concentration (554 ~g/L) at

24 hr, but deaths did not begin to occur until 48 hr at this concentration

(Table 4).' Mortalities at 96 hr were 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 in partial effects.

Adult snails (mean weight 476 Z 218 mg) were exposed to five

concentrations of nonylphenol, plus control, in duplicate 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 in the 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). No mortality occurred in the

remaining three exposures or control. The 96-hr LCSO estimate for Physella

virgata is 774 ~g/L and the 96-hr ECSO estimate is 378 ~g/L (Table 5). The

~confidence limits were not reliable because none of the test

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concentrations of nonylphenol resulted in partial mortality or effects.

Dragonfly nymphs (mean weight 285 ~ 126 mg) were exposed to five

concen~rations of nonylphenol, plus control, in duplicate 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 in the 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 in the 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. No mortality

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 2 mm in total length) were exposed to

£ive concentrations of nonylphenol, plus control, in duplicate 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) for Hyalella

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 in the 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,

11
a

there was 100% mortality in the highest concentration (264 pg/L), 85% in the

second highest concentration (160 pg/L) and 30% in the third highest

concentration (109 pg/L). No mortality occurred in the 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, in duplicate 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 in the highest (384 pg/L) concentration and within 48 hr

in the second highest (192 pg/L) concentration (Table 10). At 96 hr, there

was 100% mortality in the highest concentration (384 pg/L) and 45% in the

second highest concentration (192 pg/L). No mortality occurred in the

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,

plus control, in duplicate in flow-through exposures. Measured nonylphenol

concentrations ranged from 16.0 to 344 pg/L. Deaths began occurring within 24

hr of initiation of the test with 100\ mortality in the highest (344 pg/L)

exposure at 96 hr (Table 11). One death occurred, and one fish was affected

in the third highest (8S.1 pg/L) exposure on the last day. All of the fish in

the second highest (152 pg/L) exposure began to show effects at 48 hr after

test initiation. No mortality or effect was observed in any of the other

exposures or control. The 96-hr LC50 estimate (95% confidence limits) for

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Oncorhynchus 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, in duplicate in flow-through exposures. Hean

measured nonylphenol concentrations ranged from 109 to 2,080 ~gfL. Frond

production (new fronds) at 96 hr was significantly (a=0.05) reduced (62% of

the control) only in the highest concentration (2,080 pgfL). The remaining

exposure concentrations had frond production that was 76% or more 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 in quadruplicate. 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 (126 pgfL)

exposure. No deaths occurred in the remaining exposures or control. The 48-

hr ECSO estimate (95% confidence limits) for Daphnia magna is 84.8 (76.0-94.6)

pgfL (Table 5).

Green algae were exposed to seven concentrations of nonylphenol, plus

control in triplicate 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\

13
for 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 in both cases. Cell production (by dry weight) was 30\

and 0\ of control in the 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 in any of the

remaining exposures. The NOEC for Selenastrum capricornutum exposed to

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

mean of NOEC and the LOEC is 1,013 ~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 in Test 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 in the highest (245 ~g/L) which had

no living adults by day five. . The NOEC for D. magna exposed to nonylphenol is

14
99.5 ~g/L and the LOEC based upon survival of adults is 245 ~g/L for

Test Number . 1 and the geometric mean (chronic value) of the NOEC and the LOEC

is 156.1 ~g/L.

Dapl!nia magna «24-hr-old neonates) were exposed in Test N~er 2 to

five conclE~ntrations of nonylphenol, plus control. Measurednonylphenol

concentra1:ionsranged from 11.2 to 165 ~g/L. Nonylphenol conce~trations

decreased an average of 55.3 : 17.0\betweenrenewalof test solutions. Ten

replicate~1with one neonate per replicate were used for each exposure and

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\ in the controls at termination of exposure (Table 16).

The high mortality in the controls and lower exposures invalidated the test.

Reproduction began by day 8 at all concentrations and control, except in the

highest (165 ~g/L) exposure. The number of young produced at the termination

of exposul~e was significantly (a=0.05) different from the control only in the

highest (165 ~g/L) exposure. The NOEC for D. magna exposed to nonylphenol is

77.3 pg/L and the LOEC based upon reproduction is 165 ~g/L and the geometric

mean (chronic value) of the NOEC and the LOEC is 112.9 ~g/L.

Dapl!nia magna «24-hr-old neonates) were exposed in Test 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 in the 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 on days 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 length of survivors was

15
significantly 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) ~or this 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, in duplicate. 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 in the 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 8 days post-hatch in the third

highest (23.1 ~g/L) exposure (5 days after transfer). There was 66\ mortality

in the third highest (23.1 ~g/L) exposure by the end of the test. The

mortalities in the remaining two exposures were not significantly different

from the control. Mean standard length and wet and dry weights 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

exposed to nonylphenol is 6.0 ~g/L, and the LOEC based upon growth is 10.3

~g/L (Table 5). The geometric mean of the LOEC and the NOEC (chronic value)

for this test is 7.861 ~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. Standard guide for conducting renewal life-cycle toxicity tests

with Daphnia magna. E 1193-87. American Society for Testing and

Materials, 1916 Race Street, Philadelphia, PA. Annual Book of

Standards. Vol. 11.04. pp. 845-856.

Benoit, D.A., V.R. Mattson and D.L. Olson. 1982. A continuous-flow minidiluter system for toxicity testing. Water Res. 16:457-464.

Bhatt, B.D., J.V. Prasod, G. Kalapana and S. Ali. 1992. Separation and

characterization of isomers of p-nonylphenols by capillary GC/GC-MS/GCFTIR Techniques. J. Chromatogr. Sci. 30:203-210.

Naylor, C.G. 1993. Texaco Chemical Co., Austin, TX. (Memorandum to Larry

Brooke, University of Wisconsin-Superior, Superior, WI, January, 29.)

17
stephan, C.E., D.l. Hount, D.J. Hansen, J.H. Gentile, G.A. Chapman, and W.A.

Brungs. 1985. Guidelines for deriving 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, VA 22161. 98 pp.

18

0--
TABLE 1. Water Characteristics of Nonylphenol Exposures to Various Freshwater Organisms. Mean.:tStandard

Deviation, Range (in Parentheses) and Number of Observations (n).

Dissolved Total Alkalinity Specific

Organism & Temperature Oxygen Hardness (mg;L as pH Conductivity

Test Typel! (C) (mg/L) (mg/L as (caco) ) (llmhos/cm)

CaCo,)

Rainbow trout 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

Rainbow trout 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

Fathead minnow 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=60
TABLE 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 of Analytical Conditions Used for Analysis of Nonylphenol Toxicity Tests

Flow Rate 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 800 pg/L 1.000 275

20\ 0.1. H20, 5\ THF

Snail A/FT 40\ CH,CN, 40\ CH,OH, 1.5 No 600 pL 1.000 275

15\ 0.1. H20, 5\ THF

Dragonfly A/FT 40\ CH]CN, 40\ CH,OH, 2.5 No 600 pL 1.000 275

15\ D.I. H20, 5\ THF

Amphipod A/FT 40\ CH]CN,40\ CH)OH, 1.5 Yes 30 mL 1.000 275

20\ D.I. H2O

Fathead Minnow A/FT 40\ CH]CN, 40\ CH,OH, 1.5 No 600 pL 1.000 . 275

15\ 0.1. H20, 5\ THFY

Bluegill A/FT 40\ CH]CN, 40\ CH]OH, 2.5 No 600 pL 1.000 275

15\ 0.1. H20, 5\ THF

Rainbow Trout A/FT 95\ CH]CN or CH]OH, 1.0 No 600 pL 1.000 275

5\ D.I. HP

Rainbow Trout C/FT 40\ CH]CN, 40\ CH]OH, 1.5 Yes 10 mL 1.000 275

15\ D.I. H20, 5\ THF

Duckweed A/FT 40\ CH)CN,40\ CHpH, 1.3 No 300 pL 0.500 224

20\ D. Ie H2O

Cladoceran CjR 80\ CH]CN, 20\ D. I. H2O 1.0 No 300 pL 0.250 224

Green Algae A/S 80\ CH)CN, 20\ 0.1. Hz<> 1.0 No 300 pL 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 in Foderal Regiscer

Vol. 49, No. 209, October 26, 1984.

Two D.L. calculated due to a change in instrument sensitivity midway through

the test. .

"

TABLE 4. Dead/Affected Annelids, Lumbriculus variegacus, Exposed to Five

Concentrations of Nonylphenol Over Time. Twenty Organisms per

Exposure Concentration.

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.

TABLE 3. 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 ;t 4.1 96.1 14.7 88.0

Cladoceran A/R 94.1 3.0 90.1 ;t 7.3 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 ;t 7.8 88.7 ;t 16.4 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 ;t 12.9 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 6.8
11 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.

Geometric mean of NOEC and LOEC.

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, and wet and dry

weights.

11

23

t-

,

TABLE S. Effect Concentrations of Acute and Chronic Exposures of Nonylphenol

to Various Freshwater Organisms.

Organism 'tTestType Test Effect '-2\

05, Duration

Type Concentration (g/L)

ACUTE !rESTS

Annilid, FT (96h) LCSO 342,/ (296-396)

Lumbriculus variegatus ECSO 268/' ( '1J )

Snail, FT (96h) LCSO 774 / ( '1J )

Physella virgata ECSO 378 r ( 31 )

Dragonfly, FT (96h) EC50 596 ./ (497-714)

Ophiogomphus ap.

Amphipod, FT (96h) LCSO 20.7 : (16.8-25.6)

Hyalella azteca ECSO 20.7 (16.8-25.6)

Fathead minnow, 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 10.3
TABLE 6. Dead/Affected Snails, Physella virgata, Exposed to Five

Concentrations of Nonylphenol Over Time. Ten Organisms per Exposure Concentration.

TABLE 8. Dead/Affected Amphipods, Hyalella azteca, Exposed to Five

Concentrations of Nonylphenol Over Time. Twenty Organisms per

Exposure Concentration.

24

.-

Mean Exposure Concentrations % 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 20
TABLE 9. Dead/Affected Fathead Minnows, Pimephalespromelas,Exposedto Five

Concentrations of Nonylphenol Over Time. Twenty Organisms per

Exposure Concentration.

TABLE 10. Dead/Affected Bluegill, Lepcmis macrcchirus, Exposed to Five

COncentrations of Nonylphenol Over Time. Twenty Organisms per

Exposure Concentration.

TABLE 11. Dead/Affected Rainbow Trout, Oncorhynchus mykiss, Exposed to Pive

Concentrations of Nonylphenol Over Time. Twenty Organisms per

Exposure Concentration.

2S

Mean Exposure concentrations 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 :t S.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 20
TABLE 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 to Five Concentrations

of Nonylphenol OVer Time. Twenty Organisms per Exposure

Concentration.

26

Mean Exposure Concentrations % 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.

Mean Exposure ConcentrationsII% 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 Mean of new solution nonylphenol concentrations.
TABLE 14. Mean Growth (Dry Weight Biomass) of cultures of the

Green Algae, Selenastrum capricornutum, after 96-Hour

Exposures to Various Concentrations of Nonylphenol.

Initial Chemistry was 2 x 105 Cells per Milliliter in

100 Milliliter Test Solutions.

Significantly different from the control at aKO.OS.

27

Mean:t S.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 :t 0.4

66.7 :!:22.2 8.5 :t 0.7

136 :t 48.0 9.4 :t 1.0

329 :t:84.8 8.0 :t:1.3

694 :t:247 7.7 :!:0.8

1,480 :t 552 2.6 :t 2.48

2,720 :t 1,570 0.3 :t 0.18
TABLE 15. survival and Reproduction of the Cladoceran, Daphnia magna, Exposed

to Various Concentrations of Nonylphenol for 21 Days - Test 1.

Percent

survival of

adults

Mean number of

young/surviving

adult after 21

days

Mean nU111.ber of

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 and Reproduction of the Cladoceran, Daphnia magna, Exposed

to Various 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

survival of

adults

Mean number of 94.2 106 80.1 87.8 94.0 28.3.

young/surviving

adult after 21

days

Mean number of 88'.2 81.4 77.3 79.9 67.0 10.O.

young/starting

adult

Significantly different from control at a=0.05.
TABLE 17. Survival and Reproduction 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.

survival of

adults

Mean nW1\berof 111 113 103 118 87.5 O.

young/surviving

adult after 21

days

Mean number of 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

Significantly different from control at a-O.OS.
.-. nA~~lIaUL.~CYI ADnorma~ Development, Survival and Growth of Rainbow

Trout (Oncorhynchus mykiss) Exposed to Nonylphenol for 91 Days 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 at day 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.II 6.02.5 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

Hean percent 3.5 2.0 1.0 0.5 95.5 100

abnormal or dead

at hatchJI

Mean percent 93.3 93.3 90.0 33.351 0$1 051

survival at 51

days post-hatch

Mean wet weight 0.734 0.716 0.516$1 0.375s1 0$1 0"

(g) at 51 days

post-hatch

Mean dry weight 0.135 0.133 0.095" 0.066$1 051 OS1

(g) at 51 days

post-hatch

Mean standard 38.5 38.0 33.151 29.851 0" 0"

length (Dim) at 51

days post-hatch

Mean 1.89 1.87 1.28 0.33 0 0

biomass/chamber