Toxicity of Metals in Water and Sediment 

to Aquatic Biota

By John M. Besser and Kenneth J. Leib

Chapter E19 of

Integrated Investigations of Environmental Effects of Historical 

Mining in the Animas River Watershed, San Juan County, Colorado

Edited by Stanley E. Church, Paul von Guerard, and Susan E. Finger

Professional Paper 1651

U.S. Department of the Interior

U.S. Geological Survey
Contents

Abstract .......................................................................................................................................................839

Introduction.................................................................................................................................................839

Purpose and Scope ..........................................................................................................................840

Acknowledgments ............................................................................................................................840

Methods.......................................................................................................................................................840

Toxicity Tests with Stream Water and Sediment .........................................................................840

Toxicity Tests with Zinc and Copper ..............................................................................................842

Modeling Toxicity of Metals in Stream Water..............................................................................842

Chemical Analysis.............................................................................................................................842

Statistical Analysis ...........................................................................................................................842

Results and Discussion .............................................................................................................................842

Toxicity of Sediment .........................................................................................................................842

Toxicity of Stream Water .................................................................................................................843

Toxicity Thresholds for Zinc and Copper ......................................................................................845

Modeled Toxicity of Zinc and Copper ............................................................................................846

References Cited........................................................................................................................................848

Figures

 1. Map showing location of sampling sites for toxicity studies in Animas River 

watershed study area ..............................................................................................................841

 2–5. Graphs showing:

 2. Survival of the amphipod, Hyalella azteca, during exposure to stream water 

 and fine sediment, separately and in combination, August–September 1998 ...........843

 3. Survival of the fathead minnow, Pimephales promelas, exposed to stream 

 water, undiluted (100 percent) and in serial dilutions, August–September 1998 

 and April 1999 ......................................................................................................................844

 4. Seasonal variation in modeled dissolved zinc, hardness-adjusted toxicity 

 thresholds, and chronic water-quality criteria ..............................................................847

 5. Seasonal variation in modeled dissolved copper, hardness-adjusted toxicity 

 thresholds, and chronic water-quality criteria ..............................................................847

Tables

 1. Concentrations of metals in stream sediment, 1997 and 1998 ..........................................843

 2. Concentrations of metals in sediment pore water, August 1998 ......................................843

 3. Dissolved and total metal concentrations in stream water during toxicity tests, 

August–September 1998 and April 1999 ...............................................................................845

 4. Toxicity thresholds for copper and zinc from laboratory tests .........................................846
Abstract

We evaluated the toxicity of metals to aquatic biota of 

the upper Animas River and the lower reaches of Cement and 

Mineral Creeks based on toxicity tests with field-collected 

stream water and sediment, laboratory tests with selected 

metals of concern, and site-specific models of seasonal metal 

concentrations and metal toxicity thresholds. Stream water and 

sediment from several sites were toxic to amphipods, Hyalella 

azteca. However, stream water was less toxic than sediment, 

and the toxicity of stream water was reduced in the presence of 

sediment, presumably due to sorption of dissolved metals. The 

toxicity of stream water to amphipods and fathead minnows, 

Pimephales promelas, differed seasonally: toxicity was greater 

during late winter and early spring (before snowmelt) than in 

late spring or summer.

Toxicity of stream water was associated with elevated 

concentrations of several dissolved metals, especially zinc 

and copper. Laboratory tests demonstrated that dissolved 

zinc was highly toxic to amphipods, less toxic to fathead 

minnows, and least toxic to brook trout, Salvelinus fontinalis, 

the predominant fish species in the Animas River watershed 

study area. Significant toxic effects of zinc on early life 

stages of brook trout occurred at concentrations of 960 μg/L 

or greater. In contrast, both brook trout and fathead minnows 

were highly sensitive to copper; significant reductions in 

growth occurred at concentrations less than 10 μg/L. Models 

of the seasonal toxicity of zinc and copper in stream water 

were developed for three gauging stations near Silverton, 

Colorado. These models predicted extended periods of zinc 

toxicity to amphipods at all three sites and seasonal toxicity of copper to fathead minnows and brook trout in lower 

Mineral Creek (year-round) and in the Animas River downstream of Silverton (in winter). Predictions of these models 

were consistent with results of toxicity tests with stream 

water and with surveys of stream biotic communities, which 

suggests that dissolved copper concentrations in the study 

area are a significant limiting factor for brook trout and that 

dissolved zinc concentrations may limit populations of sensitive invertebrate taxa.

Introduction

Contamination of aquatic environments by acidic drainage and associated toxic metals is a widespread problem in 

watersheds affected by hard-rock mining (von Guerard and 

others, this volume, Chapter B). Erosion and weathering can 

mobilize high concentrations of toxic metals from exposed 

mineral deposits and altered rock, such as those occurring 

in the headwaters of Mineral Creek, Cement Creek, and the 

upper Animas River, but this process can be greatly accelerated by mining activities, which expose large amounts 

of sulfide minerals in mine tunnels, waste-rock piles, and 

deposits of mine and mill tailings (Bove and others, 2000). 

Adverse effects of acidity and associated toxic metals on 

aquatic ecosystems may extend far downstream from sources 

of acidic drainage. Fish are absent and invertebrate fauna is 

limited in highly acidic streams (pH <4.5), and adverse effects 

on aquatic communities are also severe in mixing zones 

of acidic and neutral-pH waters: examples are the reach of 

Mineral Creek downstream of the confluence with South Fork 

Mineral Creek, and the reach of the Animas River downstream 

of the confluence of Cement Creek (Besser and Brumbaugh, 

this volume, Chapter E18). Colloidal precipitates of iron 

and aluminum oxyhydroxides formed upon neutralization of 

acidic water are typically enriched with high concentrations 

of potentially toxic metals, and these precipitates may be 

transported substantial distances downstream (Kimball and 

others, 1995; Church and others, 1997; Kimball and others, 

this volume, Chapter E9). Metals may remain in bioavailable 

forms in water and sediment well downstream of mixing 

zones, leading to metal bioaccumulation and toxic effects 

on fish and invertebrates (Kemble and others, 1994; Besser, 

Brumbaugh, and others, 2001; Besser and Brumbaugh, this 

volume).

Stream biota of the Animas River watershed study area 

have been adversely affected by more than a century of mining 

activities, which came to an end in the early 1990s (Jones, 

this volume, Chapter C). Recent surveys of aquatic communities in the watershed have documented severe impairment in headwater reaches most affected by acidic drainage, 

Chapter E19

Toxicity of Metals in Water and Sediment to Aquatic Biota

By John M. Besser and Kenneth J. Leib
absence of fish in reaches below mixing zones of acidic 

tributaries, and reduced abundance of fish (principally brook 

trout) and reduced diversity and abundance of invertebrates 

in the downstream reach of the Animas River (Besser and 

Brumbaugh, this volume). In contrast, diverse and productive 

invertebrate communities and self-sustaining populations of 

brook trout occur in tributaries that are less impaired by acidic 

drainage.

Several recent studies have characterized the patterns 

of metal contamination associated with mining in the Animas 

River watershed study area. Church and others (1997) and 

Church, Fey, and Unruh (this volume, Chapter E12) have 

documented the distribution of elevated metal concentrations 

in fine bed sediment, relative to premining baseline concentrations. Leib and others (2003) and Wright, Simon, and others 

(this volume, Chapter E10) have reported that concentrations 

and loadings of metals varied in response to seasonal patterns 

of stream discharge (von Guerard and others, this volume, 

fig. 3). Concentrations of dissolved metals were generally 

lowest during the period of peak discharge in spring and 

early summer, due to dilution by melting snow, then gradually increased, starting in late summer (August–September), 

reaching annual maxima during low flow in late winter 

(March–April). Nimmo and others (1998) reported that stream 

water from Cement Creek was highly toxic to three test organisms (rainbow trout, Oncorhynchus mykiss; fathead minnow; 

and a daphnid, Ceriodaphnia dubia) in acute (48 hr) on-site 

toxicity tests conducted during August 1997 (low flow) and 

June 1998 (high flow). Patterns of metal bioaccumulation by 

fish and other components of stream food webs suggested that 

elevated concentrations of copper and zinc corresponded more 

closely than concentrations of cadmium and lead to documented effects on invertebrate and fish communities (Besser, 

Brumbaugh, and others, 2001; Besser and Brumbaugh, this 

volume).

Purpose and Scope

This chapter describes a series of studies conducted to 

better define the underlying mechanisms of observed toxicity 

to aquatic biota of the Animas River watershed and to establish reliable thresholds for recovery. The objectives of these 

studies were:

• To determine the relative toxicity of stream water and 

fine bed sediment at sites in the upper Animas River 

and major tributaries

• To determine the seasonal range of toxicity in stream 

water

• To establish site-specific thresholds for toxicity of zinc 

and copper in stream water, and

• To develop models of the contributions of copper and 

zinc to toxicity of stream water.

Acknowledgments

Ann Allert, Bill Brumbaugh, Eric Brunson, 

Doug Hardesty, James Kunz, Steve Olson, Ning Wang, and 

David Whites provided valuable assistance with field work and 

toxicity testing. Eugene Greer provided expert assistance with 

handling of brook trout embryos and fry. Jon Evans collected 

samples of stream water for toxicity testing in April 1999. 

Win Wright and Paul von Guerard provided logistical assistance during field sampling in 1997 and on-site toxicity testing 

in 1998. Del Nimmo and Janie Castle provided information 

on preliminary toxicity testing conducted in 1997. Bill Simon 

(Animas River Stakeholders Group (ARSG), Hermosa, Colo.) 

helped coordinate our efforts with other ongoing studies in 

the Animas River watershed. Dave Gerhardt (United States 

Department of Agriculture (USDA) Forest Service, Durango, 

Colo.) provided data from the 1996–97 ARSG biomonitoring 

study.

Methods

Toxicity studies focused on the conditions in the 

Animas River and the lower reaches of Cement and 

Mineral Creeks, near Silverton, Colo. (fig. 1). Samples for 

most toxicity tests were collected at four USGS streamflow 

gauging stations, three of which were located at the downstream end of the three major subbasins (A68, Animas River 

at Silverton; C48, Cement Creek at Silverton; and M34, 

Mineral Creek at Silverton); the fourth was located in the 

Animas River downstream of the confluences of these three 

streams (A72, Animas River below Silverton). During late 

summer 1998, additional samples were collected farther 

downstream on the Animas River at Elk Park (A73; about 

7 km downstream of A72). A reference site was selected 

on the South Fork Mineral Creek (SMC; located downstream of the Forest Service Campground), which supports 

a self-sustaining population of brook trout and diverse and 

productive fish and invertebrate communities.

Toxicity Tests with Stream Water and Sediment

Samples of stream water and fine bed sediment for 

toxicity tests were collected during several sampling periods between fall 1997 and spring 2002. Samples of sediment were collected from all six study sites during fall 1997. 

Samples of sediment and stream water were collected from 

five sites (excluding C48) during late summer 1998. Samples 

of stream water were collected from four sites (excluding C48 

and A73) during late winter 1999 (Besser, Allert, and others, 

2001). Additional samples of stream water were collected 

at site A72 in late winter through late spring (March–May), 

2002, to characterize toxicity before, during, and after spring 

snowmelt (Fey and others, 2002).

840 Environmental Effects of Historical Mining, Animas River Watershed, Colorado
Toxicity of Metals in Water and Sediment to Aquatic Biota 841

Figure 1. Location of sampling sites for toxicity studies in Animas River watershed study area.

Animas R vi er

Cunn ni gham

C reek

Elk Park

M

ni eral Creek

Animas

River

Cement Creek

Chattanooga Eureka

Silverton

Animas

Forks

Howardsville

Animas River

watershed 

study area

C O L O R A D O

Durango

0

0 1

1 2 MILES

2 KILOMETERS

107°50'30" 107°45' 107°37'30" 107°30'

37°

52'

30"

37°

45'

37°

58'

ElkCreek

C48

M34

SMC A68

A72

A73

South Fork Mineral Creek

Toxicity tests were conducted in general accordance 

with methods published by the U.S. Environmental Protection 

Agency (USEPA) (1994, 2000). Control and dilution water 

for these tests was a reconstituted water (hardness 113 mg/L; 

Besser, Allert, and others, 2001), with an ionic composition 

representative of late-summer conditions in the Animas River 

at Silverton (A68). Tests with fathead minnows (Pimephales 

promelas) were stocked with newly hatched larvae (less 

than 48 hr post-hatch) and lasted 7 days. Exposure chambers 

for fathead minnow tests were 1,000 mL beakers containing 250 mL of water. Tests with the amphipod, Hyalella 

azteca, were started with 7- to 14-day old individuals and 

lasted 7 days. Test chambers for amphipod tests were 300 mL 

beakers: beakers for sediment tests contained 100 mL of sediment and 150 mL of water; those for water-only tests contained 150 mL of water and a layer of fine sand. All toxicity 

tests were conducted under static-renewal conditions, with one 

replacement volume of test water added daily.
In 1997, amphipods were exposed to sediment with 

diluted well water (hardness approx. 140 mg/L as CaCO3

) 

in the laboratory. In 1998, amphipods were exposed to three 

treatments for each study site: stream water only (no sediment); sediment only (with control water); and sediment plus 

stream water. Survival of amphipods exposed to field-collected 

water and sediment were compared to survival in control water 

and control sediment (CTL, fig. 2). Control sediment was 

a formulated artificial sediment (Kemble and others, 1999; 

Besser, Allert, and others, 2001). Additional toxicity tests 

with stream water (minnows in summer 1998; minnows and 

amphipods in winter 1999 and winter/spring 2002) were conducted with stream water in a series of dilutions (100 percent, 

50 percent, and 25 percent) prepared with reconstituted water.

Toxicity Tests with Zinc and Copper

Toxicity thresholds for zinc and copper were determined 

in laboratory toxicity tests with amphipods, minnows, and 

brook trout (Salvelinus fontinalis). Tests with amphipods and 

fathead minnows were conducted following methods similar 

to those previously described. Tests with both species were 

conducted for 7 days with endpoints of survival, for both 

species, and growth (average dry weight per survivor) and 

biomass (total dry weight of survivors), for fathead minnows. 

Early-life-stage toxicity tests with brook trout were conducted 

in general accordance with guidelines published by ASTM 

(1997), using a hard reconstituted water, designed to reflect 

ambient water quality during the late fall spawning period for 

this species (Besser, Allert, and others, 2001). This test water 

was similar to the reconstituted water used for toxicity tests 

with stream water, except for greater hardness (180 mg/L as 

CaCO3

), to better match late fall water quality in the Animas 

River at Silverton. Brook trout tests spanned the eyed egg, sac 

fry, and swim-up fry life stages (71 days exposure), with endpoints of survival, growth (average dry weight), and biomass.

Modeling Toxicity of Metals in Stream Water

We modeled seasonal variation in toxicity of zinc 

and copper in stream water for three gauging stations near 

Silverton (A68, A72, and M34; fig. 1) by comparing modeled dissolved metal concentrations with site-specific toxicity 

thresholds. Seasonal variation in dissolved metal concentrations in stream water was modeled by multiple regression 

(Leib and others, 2002). Toxicity thresholds for zinc and copper, determined from laboratory tests, were adjusted to account 

for the influence of seasonal differences in water hardness on 

toxicity (USEPA, 1999; Besser, Allert, and others, 2001).

Chemical Analysis

Concentrations of metals in water and sediment were 

determined by inductively coupled plasma–mass spectroscopy 

(May and others, 1997; Besser, Allert, and others, 2001). 

Samples of stream water were analyzed for “total recoverable” 

metals (unfiltered samples; microwave digestion with nitric 

acid) and dissolved metals (filtered through 0.45 μm membrane). Pore water was collected for chemical characterization 

in 1997 and 1998, by centrifugation or with passive diffusion 

samplers (Bufflap and Allen, 1995). Filtered stream water, 

unfiltered water samples from laboratory toxicity tests, and 

pore water were analyzed without digestion. Sediment samples 

collected in 1997 were subjected to a partial extraction (dilute 

HCl and hydrogen peroxide; Church, Fey, and Unruh, this 

volume). Samples collected in 1998 were analyzed for total 

recoverable metals. Water samples were analyzed for iron, 

zinc, copper, cadmium, and lead; sediment samples were analyzed for these metals and aluminum.

Statistical Analysis

Data were transformed to improve normality and homogeneity of variance. Analysis of variance (ANOVA) was performed with SAS/STAT software (version 8.2; SAS Institute, 

Cary, N.C.). Concentration-response curves from toxicity 

tests were analyzed with ToxStat software (version 3.5; 

Lincoln Research Associates, Bisbee, Ariz.). Median lethal 

concentrations (LC50; for survival endpoint) or median effect 

concentrations (EC50; for other endpoints) were determined 

by the trimmed Spearman-Karber method (Hamilton and 

others, 1977). For toxicity tests with zinc and copper, lowestobserved-adverse-effect concentrations (LOECs; the lowest 

exposure concentration causing statistically significant adverse 

effect) were determined based on ANOVA, and concentrations 

associated with 25 percent inhibition of survival or growth 

(IC25s) were estimated by a linear interpolation technique 

(USEPA, 1994).

Results and Discussion

Toxicity of Sediment

Stream sediments from the Animas River watershed 

study area were not highly toxic to the amphipod, Hyalella 

azteca. Sediment toxicity tests conducted in late summer 1997 

showed little evidence of toxicity to amphipods, except for 

slight reductions in growth of amphipods exposed to sediment 

from site C48 in lower Cement Creek (Besser, Allert, and 

others, 2001). In tests conducted in summer 1998, survival 

of amphipods in the artificial control sediment was low 

(50 percent), but amphipod survival was high (>80 percent) 

in four of the five field-collected sediments (fig. 2). Survival 

of amphipods exposed to all field-collected sediments was 

greater than that in the control, but survival of amphipods in 

sediment from A72 was significantly less than in sediment 

from the reference site, SMC.

842 Environmental Effects of Historical Mining, Animas River Watershed, Colorado
Toxicity of Metals in Water and Sediment to Aquatic Biota 843

Concentrations of most metals in stream sediment 

and pore water did not correspond closely to observed toxic 

effects on amphipods. Sediments from C48 and A72, both of 

which were toxic in one of the two years, contained greatest 

concentrations of sediment iron, but relatively low concentrations of other metals (table 1). Pore water from C48 sediment 

was acidic and contained highest concentrations of all metals analyzed in 1997 (Besser, Allert, and others, 2001), but 

greatest concentrations of iron and other metals in pore water 

were in sediments from A68 or M34, which were not toxic 

(tables 1 and 2).

Toxicity of Stream Water

Toxicity of stream water to amphipods and fathead 

minnows followed different spatial and temporal patterns. 

Stream water was highly toxic to amphipods in all seasons 

tested. In summer 1998, survival of amphipods in stream water 

from all five sites was significantly reduced relative to survival 

in the control water (fig. 2). Mortality of amphipods in site 

water ranged from 94 percent to 100 percent. Survival of 

amphipods was generally greater in tests with both stream 

water and sediment than in those with stream water only, suggesting that stream sediment reduced the toxicity of stream 

water by sorption of dissolved metals. Undiluted stream water 

from A68, A72, and M34 also caused 100 percent mortality of amphipods during late-winter low-flow conditions in 

April 1999 (Besser, Allert, and others, 2001), and water from 

EXPLANATION

Exposure type

Water

Water+sediment

Sediment

Figure 2. Survival of the amphipod, Hyalella azteca, during 

exposure to stream water and fine sediment, separately and in 

combination, August–September 1998. Means, with standard 

errors, n=6 per treatment. Asterisks indicate significant reduction 

in survival relative to corresponding control group (ANOVA/

Dunnett’s test).

Table 1. Concentrations of metals in stream sediment, 1997 and 1998. 

[For each site and metal, first value is result of partial extraction (1N HCl, 1 percent H2O2), September 1997, and second value is result of total-recoverable 

extraction, August 1998; n=1; -- indicates no sample]

Site Aluminum Iron Cadmium Copper Lead Zinc

(percent dry weight) (micrograms per gram dry weight)

A68 0.44 / 1.0 1.4 / 2.6 11 / 13 265 / 372 1,740 / 1,790 2,030 / 2,640

A72 0.47 / 1.9 2.5 / 8.4 3.7 / 7.0 175 / 447 782 / 782 676 / 1,010

A73 0.44 / 1.2 2.2 / 4.9 4.1 / 6.8 174 / 480 725 / 725 830 / 815

SMC 0.39 / 1.0 0.87 / 2.6 2.1 / 1.1 21 / 19 246 / 246 531 / 94

M34 0.53 / 1.5 2.5 / 4.7 1.2 / 2.6 148 / 156 221 / 221 349 / 196

C48 0.36 / -- 3.8 / -- 0.27 / -- 42 / -- 278 / -- 94 / --

Table 2. Concentrations of metals in sediment pore water, August 1998.

[Metal concentrations in micrograms per liter; means (n=4), with standard error in parentheses. For each metal, means with the same superscript letter are not 

significantly different (analysis of variance, with Duncan’s multiple-range test)]

Site Aluminum Iron Cadmium Copper Lead Zinc

A68 871 (688)a 1,610 (1,253)b 9.3 (0.9)a 60 (34)a 165 (111)a 946 (261)a

A72 204 (70)a 8,180 (315)a 0.09 (0.04)d 5.3 (2.5)b 7.6 (3.7)b 42 (5.2)c

A73 121 (40)a 298 (99)b 3.5 (0.1)b 13 (1.6)ab 4.0 (1.1)b 167 (5.6)b

SMC 218 (54)a 154 (61)b 0.86 (0.24)c 9.1 (5.7)b 5.5 (1.6)b 63 (45)c

M34 137 (56)a 34,200 (675)a 0.07 (0.02)d 4.5 (1.1)b 2.6 (0.9)b 56 (3.0)c
A72 caused 98–100 percent mortality of amphipods in three 

tests conducted before, during, and after spring snowmelt in 

2002 (Fey and others, 2002). In contrast, stream water from 

most sites tested showed seasonal differences in toxicity to 

fathead minnows. In summer 1998, survival of minnows 

exposed to stream water was significantly reduced, relative to 

survival in control water, by undiluted stream water from only 

one site (SMC) of the five tested. In contrast, undiluted stream 

water from three of the four sites tested in late winter 1999 

(M34, A72, and SMC) caused significant reductions in survival 

(fig. 3). Survival of minnows was unaffected by water from 

A68 during both seasons.

Differences in the toxicity of stream water among sites, 

test organisms, and sampling periods reflected overall trends 

in dissolved and total metal concentrations in stream water. 

Stream water from the most toxic site, M34, contained greatest concentrations of dissolved and total aluminum, iron, and 

copper (table 3). Water from A72, which was also toxic to 

both minnows and amphipods, contained a similar mixture 

of metals at somewhat lower concentrations. Attenuation of 

metal concentrations is evident at the downstream site A73, 

which was toxic to amphipods but not to fathead minnows in 

summer 1998. In addition, the relative toxicity of stream water 

to amphipods and minnows differed among sites. Water from 

A68, which was highly toxic to amphipods but not to minnows, 

had consistently high concentrations of dissolved and total zinc 

and cadmium, and lesser concentrations of copper, aluminum, 

and iron (table 3). In contrast, sites that were most toxic to minnows (A72 and M34) had consistently high concentrations of 

copper and iron and lower concentrations of zinc. The greater 

toxicity of stream water at A72 and M34 to fathead minnows in 

spring 1999, relative to summer 1998, was also associated with 

greater concentrations of iron and copper. The toxicity of water 

from SMC to minnows and amphipods during summer 1998 

was associated with transient elevated concentrations of several 

metals, notably aluminum, apparently reflecting the influence 

of a localized thunderstorm during this test (Besser, Allert, and 

others, 2001). Although the South Fork Mineral Creek watershed has had a low level of mining activity compared to the 

rest of the study area, there is at least one large inactive mine 

upstream from this study site that could cause periodic impairment of water quality (Bandora mine, site # 332; Church, Mast, 

and others, this volume, Chapter E5).

Dissolved and (or) total concentrations of several metals 

in stream water frequently exceeded national chronic waterquality criteria (WQC) at the sites studied (USEPA, 1999; 

table 3). Hazards of toxicity due to dissolved zinc and copper 

were strongly suggested by numerous measured concentrations 

exceeding WQC values, and differences in toxicity among 

seasons and between species were consistent with variation 

in concentrations of these two metals. Concentrations of both 

aluminum and iron also frequently exceeded WQC values at 

all sites except A68. However, the potential toxic effects of 

waterborne aluminum and iron at these sites are uncertain. 

Relatively few studies have evaluated the toxicity of these 

metals, and most of these studies were conducted with dilute 

test water that differed substantially from the typical water 

quality of the study watershed. The greatest hazard of toxicity for these metals is associated with mixing zones of acidic 

and neutral water. The toxicity of aluminum to brook trout is 

generally thought to be greatest at pH 5.0–5.5, with toxicity 

thresholds in the range 300–400 μg/L (for example, Cleveland 

and others, 1986; Mount and others, 1988). However, toxicity 

of aluminum has been shown to be ameliorated by increased 

calcium concentrations, and most studies of aluminum toxicity have been conducted in water with calcium concentrations 

much lower than those typical of the Animas River watershed 

study area. The toxicity of aluminum in mixing zones seems 

to be associated with formation of oxyhydroxide colloids, but 

A

B

EXPLANATION

Percent stream water

Control

25 percent

50 percent

100 percent

Figure 3. Survival of the fathead minnow, Pimephales promelas, 

during exposures to stream water and dilutions. A, August–

September 1998; B, April 1999. Means, with standard errors; 

n=3 per treatment. Asterisks indicate significant reduction in 

survival relative to corresponding control group (ANOVA/Dunnett’s 

test).

844 Environmental Effects of Historical Mining, Animas River Watershed, Colorado
Toxicity of Metals in Water and Sediment to Aquatic Biota 845

these effects tend to be very localized as toxicity of newly 

formed colloids decreases rapidly (Verbost and others, 1995). 

Toxicity data for iron are also limited, and effects of dissolved 

iron on brook trout have been reported to vary widely under 

different test conditions. Toxicity of particulate iron to brook 

trout is low, and standard method for separation of dissolved 

metals (0.45 μm filtration) does not adequately separate iron 

colloids from truly dissolved metal iron (Church and others, 

1997; Kimball and others, this volume). Measurements of 

filtered iron concentrations therefore tend to overestimate concentrations of dissolved iron. Toxicity of iron in stream water 

may be best represented by concentration of dissolved ferrous 

ions (Fe+2), the predominant dissolved iron species in highly 

acidic streams, such as Cement Creek and upper Mineral 

Creek. Available data suggest that hazards of iron toxicity in 

the Animas River watershed study area are also associated with 

elevated concentrations of dissolved iron under slightly acidic 

conditions, such as winter conditions at A72 and M34 (table 3).

Toxicity Thresholds for Zinc and Copper

Toxicity thresholds determined from laboratory tests with 

zinc and copper are consistent with the observed differences in 

toxicity of stream water among species and seasons. Zinc was 

more toxic to amphipods than to fathead minnows, but copper 

was more toxic to minnows than to amphipods (table 4). These 

differences are consistent with the responses of these species 

to stream water, as water from A68 (highest zinc, low copper) 

was not toxic to minnows, whereas water from M34 (highest 

copper, low zinc) was highly toxic to minnows (figs. 2 and 

3). The effect of seasonal variation in metal concentration is 

evident from repeated tests with fathead minnows exposed to 

stream water from A72, which was not toxic during late spring 

and late summer (1998 and 2002), but was highly toxic during 

late winter and early spring (1999 and 2002), when copper concentrations approached the LC50 for this species.

Toxicity tests with brook trout established chronic toxicity thresholds for the early life stages that are present in the 

Animas River watershed study area during winter low-flow 

conditions. Brook trout spawn in late fall, but hatching probably 

occurs during winter or early spring, the period of lowest stream 

flow and highest dissolved metal concentrations (Leib and 

others, 2003). Brook trout, like fathead minnows, were more 

sensitive to copper than to zinc (table 4). No effects on survival, 

and less than 25 percent reduction in growth, were observed 

at the highest zinc concentration tested (2,000 μg/L). The low 

sensitivity of brook trout to zinc in this test, which was conducted in hard water (180 mg/L), contrasts with tests conducted 

in softer water. A previous study found that early life stages of 

brook trout were more sensitive to zinc in soft water (LOEC 

for survival=1,368 μg/L at a hardness of 45 mg/L; Holcombe 

and others, 1979). Published thresholds for chronic toxicity of 

copper are more similar between tests with different test waters. 

A previous chronic study with brook trout in soft water found 

LOECs for survival and growth (17.5 μg Cu/L at hardness of 

45 mg/L; McKim and Benoit, 1971) that were similar to those 

from our test with hard water (LOECs from 6 to 25 μg Cu/L; 

table 4). Additional short-term (7 day) toxicity tests conducted 

in our laboratory support the hypothesis that hardness, especially calcium, has a greater influence on the toxicity of zinc 

than on the toxicity of copper (Besser, Allert, and others, 2001).

Toxicity thresholds determined from our laboratory tests 

indicate that the organisms used for testing the seasonal toxicity of stream water were appropriate surrogates for determining conditions that can be tolerated by resident aquatic biota 

of the Animas River watershed (table 4). Thresholds for 

copper toxicity were similar for fathead minnows and brook 

trout. The threshold for toxic effects of zinc on fathead minnows provides a substantial margin of safety for brook trout. 

Amphipods are much more sensitive to zinc than brook trout, 

and the available toxicity literature suggests that the threshold 

for zinc toxicity to amphipods should protect a large proportion of aquatic invertebrate taxa (USEPA, 1999).

Table 3. Dissolved and total metal concentrations in stream water during toxicity tests, August–September 1998 and April 1999.

[Metal concentrations in micrograms per liter; dissolved (filtered) samples are shown in italic bold; means, n=2–5 for 1998, n=2 for 1999. Water-quality criteria 

(WQC; USEPA, 1999) are listed for hardness of 100 mg/L for 1998 and 200 mg/L for 1999, except criteria for Al and Fe are independent of hardness; nd, not 

detected; --, no sample]

Site Aluminum Iron Zinc Copper Lead Cadmium

1998 1999 1998 1999 1998 1999 1998 1999 1998 1999 1998 1999

A68 43

77 --

94

637

nd

201

275

290

659

613

6.4

8.3

2.3

8.4

0.71

3.7 

nd

3.0

1.1

1.1

2.1

2.1

A72 42

1,129 --

402

1,622

2,150

4,805

175

240

625

574

5.9

19.2

8.3

27

<0.13

5.0 

nd

9.2

0.73

0.90

2.3

2.1

A73 61

1,062 --

151

1,420 --

178

265 --

4.7

18.4 --

<0.47

6.5 --

0.80

0.96 --

SMC 43

363 --

355

502

414

1,495

13

31

7

26

3.8

4.7

nd

1.2

0.42

3.6 

nd

2.2

0.10

0.18

0.58

0.43

M34 333

1,382 --

414

2,020

3,315

5,605

210

180

397

347

6.7

24.8

23

37

0.11

5.4 

<0.3

9.6

0.73

0.75

1.9

1.4

WQC 87 1,000 120 216 9.3 16.9 3.2 7.7 2.5 4.2
Modeled Toxicity of Zinc and Copper

Seasonal variation in toxicity of stream water of the 

Animas River watershed reflects seasonal variation in concentrations of dissolved zinc and copper, relative to site-specific 

toxicity thresholds for these metals. Concentrations of zinc 

and copper typically followed similar seasonal patterns at 

the three gauging stations near Silverton (fig. 1), with lowest 

concentrations in early summer and greatest concentrations in 

late winter (fig. 4; Leib and others, 2003). The only exception 

was dissolved copper at site A68, Animas River at Silverton, 

which remained at very low concentrations during the entire 

low-flow period (summer through early spring) but showed a 

moderate increase during high flows in late spring. Thresholds 

for toxicity of zinc and copper to brook trout and amphipods 

(table 4), adjusted for seasonal variation in hardness using the 

regressions published by USEPA (1999; Besser, Allert, and 

others, 2001), followed similar seasonal trends (figs. 4 and 

5). Modeled thresholds for toxicity of copper and zinc at all 

three sites reached minima in late spring, corresponding to 

the annual minimum for water hardness.

Greatest toxicity of zinc in stream water was predicted 

for site A68, where dissolved zinc concentrations exceeded 

hardness-adjusted toxicity thresholds for all three species 

during late winter and early spring (fig. 4). Stream water from 

all three sites greatly exceeded both the chronic water-quality 

criteria for zinc and the threshold for toxic effects of zinc on 

survival of amphipods year-round (table 4; USEPA, 1999), 

consistent with the findings of our toxicity tests. Only stream 

water at A68 approached the threshold for chronic toxicity of 

zinc on brook trout. These predictions are consistent with the 

absence of toxic effects in fathead minnows exposed to water 

from A68 and A72 during late spring and summer. The lesser 

zinc toxicity predicted for site M34 suggests that reduced 

survival of fathead minnows in stream water from M34 during 

both summer and winter cannot be solely attributed to zinc 

toxicity.

Modeled toxicity of copper in stream water followed different patterns among the three sites (fig. 5). Dissolved copper 

concentrations were greater and had more seasonal variability 

at both M34 and A72. Concentrations of dissolved copper at 

A68 were below toxicity thresholds and water-quality criteria year-round, except for a brief period during early spring 

snowmelt. Modeled copper concentrations in stream water 

exceeded toxicity thresholds for brook trout year-round at 

M34, during winter and spring at A73, and during late spring 

at A68. Because the threshold for chronic toxicity of copper 

to brook trout may not be strongly influenced by hardness, 

the severity of the risks associated with exceeding toxicity 

thresholds for copper may differ among the three sites. Toxic 

effects were predicted for brook trout at A72 and M34 during 

periods of high water hardness (greater than that in toxicity 

tests), when hardness-adjusted toxicity thresholds may underestimate actual toxicity hazards. In contrast, dissolved copper 

concentrations at A68 exceeded toxicity thresholds for brook 

trout only during snowmelt, the annual minimum for hardness, 

when hardness-adjusted toxicity threshold may over-predict 

toxicity.

Toxic units, ratios of modeled metal concentrations to 

hardness-adjusted toxicity thresholds, indicated greater hazards of zinc toxicity to amphipods and greater hazards of copper toxicity to both fish species. Toxic units of 1.0 and greater 

indicate that dissolved metal concentrations exceed hardness 

adjusted toxicity thresholds (Besser, Allert, and others, 2001). 

The sum of toxic units for copper and zinc, an estimate of 

potential additive toxicity of these metals, was greater during late winter than during the late summer, consistent with 

observed seasonal differences in toxicity. Cumulative hazards 

to amphipods were greatest at A68, primarily due to high zinc 

concentrations, and hazards to trout were greatest at M34, 

primarily due to high copper concentrations. Hazards to trout, 

estimated from cumulative toxic units, were only slightly 

greater at A72, where trout are absent, than at A68, where 

both adult trout and juveniles have been collected in recent 

Table 4. Toxicity thresholds for copper and zinc from laboratory tests.

[Hardness of test water=113 μg/L. LOEC, lowest observed effect concentration; IC25, 25 percent inhibition concentration; EC50, median (50 percent) effect 

concentration. See text and Besser, Allert, and others (2001) for derivation of toxicity thresholds]

Species Endpoint

Copper 

(μg/L)

Zinc 

(μg/L)

LOEC IC25 EC50 LOEC IC25 EC50

Amphipod Survival 100 64 79 250 183 220

(H. azteca)

Fathead minnow Survival 12.5 17 35 500 458 704

(P. promelas)

Growth 100 9.2 55 >1,000 >1,000 >1,000

Biomass 50 8.7 11.4 500 >1,000 >1,000

Brook trout Survival 25 24 29 >2,000 >2,000 >2,000

(S. fontinalis)

Growth 6.3 8.1 17 1,000 >2,000 >2,000

Biomass 6.3 9.8 17 >2,000 >2,000 >2,000

846 Environmental Effects of Historical Mining, Animas River Watershed, Colorado
Toxicity of Metals in Water and Sediment to Aquatic Biota 847

Figure 4. Seasonal variation in modeled dissolved zinc, 

hardness-adjusted toxicity thresholds, and chronic water-quality 

criteria. Dissolved zinc and hardness modeled as described by 

Leib and others (this volume). Toxicity thresholds for trout and 

amphipods adjusted for variation in hardness using the slope 

from the chronic water-quality criterion equation (USEPA, 2002).

A68

A72

M34

EXPLANATION

Dissolved zinc

Water-quality criterion

Trout growth

Amphipod survival

DISSOLVED ZINC, IN MICROGRAMS PER LITER

Figure 5. Seasonal variation in modeled dissolved copper, 

hardness-adjusted toxicity thresholds, and chronic water-quality 

criteria. Dissolved copper and hardness modeled as described 

by Leib and others (this volume). Toxicity thresholds for trout and 

amphipods adjusted for variation in hardness using the slope 

from the chronic water-quality criterion equation (USEPA, 2002).

EXPLANATION

Dissolved copper

Water-quality criterion

Trout growth

Amphipod survival

A68

A72

M34

DISSOLVED COPPER, IN MICROGRAMS PER LITER
years. However, hazards at A72 were predominantly due to 

copper, where toxic units for copper averaged greater than 1.0 

on an annual basis, whereas both copper and zinc contributed 

equally to cumulative toxic units for trout at A68, and neither 

individual metal had toxic units greater than 1.0 on an annual 

basis.

Both toxicity tests with stream water and site-specific 

toxicity models indicated that toxicity of zinc and copper contributes to adverse effects on aquatic biota of the Animas River 

watershed study area. Observed and predicted toxic effects of 

stream water corresponded closely to impairment of stream 

biotic communities. Toxicity of stream water was greatest at 

sites where abundance and taxonomic richness of fish and 

invertebrate communities are most impaired (M34 and A72), 

intermediate at sites with moderate impairment (A68, A73), 

and lowest at sites with the most diverse and abundant biotic 

communities (SMC). Modeled toxicity, based on ambient 

metal concentrations and site-specific toxicity thresholds, suggests that populations of brook trout in streams of the Animas 

River watershed study area are probably affected by toxic 

effects of copper but not zinc.

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