ecological archives m080-014-a1 mari k. reeves

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Ecological Archives M080-014-A1

Mari K. Reeves, Peter Jensen, Christine L. Dolph,

Marcel Holyoak, and Kimberly A. Trust. 2010.Multiple stressors and the cause of amphibian

abnormalities.Ecology 80:423440.

Appendix A. Supplemental methodological information for

contaminants sampling, data reduction, and the toxicity experiment.

Contaminants Sampling and Analysis

Two sediment samples were collected from each pond, one for

organic contaminant analysis and one for inorganic contaminant

analysis, using the methods of Csuros (1994). Samples were

homogenates pooled from three random locations in a pond. At each

location, we sampled the top 2030 cm of sediment. Shallow site

samples were collected with hand-held scoops stainless steel for

organics and plastic for inorganics. Deeper sites were sampled with

an Eckman dredge. Organic samples were homogenized in stainless

steel bowls. Inorganic samples were homogenized in Ziploc bags.

Prior to sampling each site, equipment was decontaminated by

washing with Alconox and water, rinsing with deionized water

followed by hexane and then acetone. Inorganic samples were

analyzed at the Trace Element Research Lab (TERL) in College

Station, Texas. Organic samples were analyzed at the Geochemical

and Environmental Research Group (GERG) in College Station,Texas. Sample results were compared to sediment toxicity

thresholds presented in the National Oceanic and Atmospheric

Administration, Screening Quick Reference Tables (Buchman

2008). Water samples were also collected from study ponds in 2004

and 2005 to sample for total inorganic elements using standard field


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collection protocols (Csuros 1994) and inductively coupled plasma/

mass spectrometry at TERL. Sample results were compared to water

quality criteria presented in the National Oceanic and Atmospheric

Administration, Screening Quick Reference Tables (Buchman

2008). The contaminants we measured (metals and chlorinated

organic pollutants) should remain relatively consistent through time,

with the exception of some of the lighter molecular weight aromatic

compounds.

Reducing the complexity of the contaminants data

After screening contaminants data for toxicants above at least one

established toxicity threshold, we used principal components

analyses (PCA) to reduce the dimensionality of contaminants data

separately for organic and inorganic contaminants. These groups

were kept separate because organic and inorganic compounds may

have different environmental sources, different environmental fate

and transport, and different modes of toxicity. If a contaminant was

not detected at a site, half the detection limit for that compound was

used as a substitute.

Inorganic contaminants exceeding at least one toxicity threshold in

water were aluminum, barium, cadmium, copper, iron and

manganese. Elements exceeding at least one threshold in sediment

included arsenic, cadmium, copper, iron, manganese, nickel, and

zinc. Cu was only detected in water from one site, and was therefore

excluded from the PCA. All elements in water and sediment arsenic

were log transformed to improve linearity prior to PCA. Theseelements were then subjected to PCA to result in the inorganic

vectors.

Organic contaminants exceeding at least one toxicity threshold

included phenanthrene (a polycyclic aromatic hydrocarbon or PAH),


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polychlorinated biphenyls (PCBs) and the following organochlorine

pesticides: aldrin, mirex, heptachlor-epoxide,

dichlorodiphenyltrichloroethane (p,p-DDT and metabolites),

lindane (BHC and metabolites), and chlordane (and metabolites

See Appendix B: Table B1 for metabolites detected). For the

pesticides, parent compounds and metabolites were summed prior to

PCA because they were correlated and because this made data

interpretation more straightforward.

For the metals, PCA vector 1 explained 33% of the variance and was

positively correlated (r0.5) with the following elements: Iron,

Manganese, and Nickel in sediment and Aluminum, Barium, Iron,and Manganese, in water. This vector was also negatively correlated

(r -0.5) with sediment Arsenic and Cadmium (for correlations, see

Table A1). PCA vector 2 explained 25% of the variance and was

positively correlated (r 0.5) with Copper, Iron, Nickel, and Zinc in

sediment. The third vector explained an additional 15% of the

variance, but was redundant with the first two vectors and was

therefore not retained for analysis. These PCA vectors were then

used to represent the metals with which they were correlated in theregression analysis.

The organic data required several manipulations before PCA. First,

one site was excluded from the organic PCA (and from all statistical

analyses) because the sample was taken during a forest fire and

concentrations of organic contaminants at this site were

approximately an order of magnitude higher than all other sites,

probably due to mobilization of these compounds by the fire anddeposition in ash (Site KNA60; See Appendix B; Table B3 for data).

Second, several organic contaminants representing a parent

compound (e.g., DDT) and its metabolites (e.g., DDD and DDE)

were quantified and reported separately by the analytical lab. We
http://www.esapubs.org/archive/mono/M080/014/appendix-B.htmhttp://www.esapubs.org/archive/mono/M080/014/appendix-B.htmhttp://www.esapubs.org/archive/mono/M080/014/appendix-B.htm


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summed these chemicals (parent compounds and metabolites of

DDT, chlordane, and lindane) before the PCA because we believed

they would have similar environmental fate, transport, and relatively

similar toxicological effects. Additionally, this step eased data

interpretation. After these manipulations, we included the following

organic contaminants which were over at least one toxicity threshold

in at least one study site in the PCA: the PAH phenanthrene, total

PCBs, and the organochlorine pesticides aldrin, heptachlor-epoxide,

mirex, lindane (and metabolites benzene hexachloride or BHC),

chlordane (and metabolites), and DDT (and metabolites DDD and

DDE). We performed PCA on these untransformed variables. This

resulted in the four components presented in Table A2. The firstcomponent explained 38% of the variance and was positively

correlated (r0.5) with the following compounds: total PCBs, aldrin,

heptachlor-epoxide, mirex, and chlordane. The second vector

explained an additional 25% of the variance and was positively

correlated (r 0.5) with lindane and DDT and negatively correlated

(r -0.5) with total PCBs (Table A2). The third vector explained an

additional 12% of the variance, but was only correlated with

heptachlor-epoxide, which was also correlated with the first vector,and was therefore not retained for analysis. We therefore retained

the first two PCA vectors to represent organic contaminants in the

statistical analysis.

Site Sediment and Water Exposure Experiment

Sediments were collected from six sites in late April with hand-held

stainless-steel scoops or Eckman dredge. Sediment samples werecomposites of three locations in a pond, homogenized in stainless

steel buckets. Sampling equipment was decontaminated between

sites by washing with Alconox and water, rinsing with DI water,

then rinsing with hexane, then acetone, to remove organic


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contaminants and prevent cross-contamination between sites.

Sediments were sorted to remove predatory invertebrates. On 12

May 2006, we collected six amplecting pairs of wood frogs from

two breeding sites at which abnormalities have consistently been

found and allowed them to oviposit in glass bowls. After

oviposition, adults were released at their breeding sites and extra

eggs were returned to the egg mass cluster. We collected site water

twice per week with certified chemically-clean, 5-gallon cubitainers

(Hedwin Corporation, Baltimore, Maryland, USA). The same

cubitainer was used to collect water from each site at each water

change. Water was changed every 46 days to prevent tadpoles from

fouling the water. Old water was drained with site-dedicated siphonhoses, taking care to not harm the tadpole or disturb the sediment. It

was replaced with temperature-equilibrated site water collected

either that day or the day before. Water changes began after eggs

hatched. Once tadpoles were free-swimming (Gosner (1960) stage

20), they were fed NASCO frog brittle for tadpoleXenopus, ad

libitum at each water change. Four blocks, water baths with 24

bowls each, controlled the temperature of experimental units which

were kept indoors under full-spectrum lighting on a light cyclesimulating field conditions. Temperature was also set to mimic

surface temperatures recorded in the field.


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FIG. A1. Map of Kenai Study Sites (). Heavy black lines are

paved roads. Lighter black lines are gravel roads. Dark gray shading

is KNWR boundary. Light gray shading is designated wilderness.

TABLE A1. Correlations between Metals PCA vectors and elements

that exceeded toxic thresholds in sediment and water.


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TABLE A2. Correlations between Organic PCA vectors and

chemicals that exceeded toxic thresholds in sediment and water.

TABLE A3. Table of pairwise correlations between predictor

variables used to model skeletal and eye abnormalities in wood

frogs.


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LITERATURE CITED

Buchman, M. F. 2008. NOAA Screening Quick Reference Tables.

NOAA OR&R Report 08-1, Seattle, Washington. Office of

Response and Restoration Division, National Oceanic and

Atmospheric Administration, 34 pp.

Csuros, M. 1994. Environmental sampling and analysis for

technicians. CRC Press. Boca Raton, Florida, USA.

ecological archives m080-014-a1 mari k. reeves

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