The Physiological Effects of

Nickel Chloride Hexahydrateon

Aquatic Microbial Biofilm

Presented by: Desiree’ Shaw

Mentored by: Dr. Sally Entrekin

Adam Musto

Background at http://faculty.uca.edu/sentrekin/

Aquatic Microbial Biofilm

• Complex communities of aquatic microorganismshttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC99016/

• Bacteria

• Fungi

• Important foundation of aquatic food web

• Eaten by benthic macroinvertebratesftp://ftp.wcc.nrcs.usda.gov/wntsc/strmRest/wshedCondition/EPTIndex.pdf

• Ephemeroptera

• Plecoptera

• Trichoptera

Aquatic Food Chain

Nickel

• Common metal found in surface watersOffice of Water, Regulations and Standards, Criteria and Standards Division. United States Environmental Protection Agency

• Naturally found in Earth’s crusthttp://www.atsdr.cdc.gov/toxprofiles/tp15-c6.pdf

• Released into the environment• Dust in windstorms

• Eruption of volcanoes

• Contamination of ground and surface waterhttp://www.atsdr.cdc.gov/toxprofiles/tp15-c6.pdf

• By-product of many industrial processes• Industrial waste enter environment through water cycle

• Natural gas hydraulic fracturing drilling

Natural Gas Hydraulic

Fracturing Drilling

• Fayetteville Shale

Gas Region• Highlighted portion

• Scott Henderson

Gulf Mountain Wildlife

Management Area• Blue arrow

http://www.searcychamber.com/economic-development/fayetteville-shale

Question

• This study attempted to • expand on existing data to explore the

physiological effects of nickel on the aquatic microbial community (fungi and bacteria) as measured by respiration

• identify the physiological response of the biofilm, which are a food source for the macroinvertebrates, in order to provide further understanding of the macroinvertebrate response to elevated metals

Hypothesis

• It was hypothesized that:

• aquatic microbial biofilms require trace amounts of nickel to enhance respiratory functions

• as nickel concentrations increase, the microbial community would be adversely affected, and less oxygen would be consumed by the aquatic microbial biofilm

Sweet Gum Leaf Preparation

• Cut 105 Sweet Gum (Liquidambar styraciflua) leaves into 2 cm by 2 cm squares

• Leeched leaves in hot tap water for 12 hours

Culturing of Aquatic Biofilm

• Incubated leaves in enriched water for 25 days at room temperature

• Leaves placed in mesh bag in plastic tank for containment

• Bubblier added to provide oxygen

• Enriched water changed once per week• 1400 μg/L Nitrate

• 140 μg/L Phosphorus

• 2 liter of distilled water

Incubation of Leaves

Leaves in incubation tank after 25 days

Leaves after incubation period (visible dark spots are biofilm)

Nickel Classification Criteria

• EPA established criteriahttp://water.epa.gov/scitech/swguidance/standards/criteria/current/index.cfm

• Chronic

• Acceptable

• Actual nickel levels acquired from Scott Henderson Gulf Mountain Wildlife Management Area streams• Black Fork Stream

• Clifty Stream

• Sunny Side Stream

Nickel Criteria, Classification,and Concentrations

Classification CriteriaConcentration of

Nickel (mg/L)

Blank No nickel nor leaves 0.00

Control No nickel 0.00

EPA Chronic EPA assignment 0.05

Low Black Fork Stream 0.20

Medium Clifty Stream 0.70

EPA Acceptable Allowed value under EPA standards 1.10

High Sunny Side Stream 1.60

Elevated Comparison value 3.00

Nickel Solution

• Formulate 3 mg Ni2+/L

• Nickel source from nickel chloride hexahydrate(NiCl2*6H2O)

• Ni2+ is 24.7% of the weight of NiCl2*6H2O (237.71 g/mol)

• Mass needed is calculated

• 0.003 g Ni2+ needed/0.247 percent Ni2+ in NiCl2*6H2O

• 0.01214 g NiCl2*6H2O needed for this concentration

• 0.0122 g of nickel chloride hexahydrate weighed out

• Dissolved in 1 liter enriched water

Dilution of Nickel Concentrations

Classification

Concentration

of Nickel

(mg/L)

Volume Nickel for

dilution into 500 mL

solution (mL)

Volume Nutrient water

for dilution to 500 mL

solution (mL)

Blank 0.00 0.00 500.00

Control 0.00 0.00 500.00

EPA Chronic 0.05 8.33 491.67

Low 0.20 33.33 466.67

Medium 0.70 116.67 383.33

EPA Acceptable 1.10 183.33 316.67

High 1.60 266.67 233.33

Extreme 3.00 500.00 0.00

Sample Preparation

• Twenty-four sterile 50 mL centrifuge tubes

• Fifteen incubated leaves per nickel classification

• Eight 500 mL beakers

Dissolved Oxygen Data

• Calibrated Dissolved Oxygen (DO) probe

• Initial DO levels recorded

• Incubated leaves with biofilm for 24 hours in nickel solution in the dark

• Final DO levels recorded

Biofilm Weight

Dry weight of incubated leaves determined

Ash weight of incubated leaves determined

Change in Dissolved Oxygen

Relationship of concentrations of nickel in terms

of change in dissolved oxygen per gram

± Standard Error

0.00

50.00

100.00

150.00

200.00

250.00

% S

atu

rati

on

/ gr

am

Nickel Concentrations

Control

EPA Chronic

Low

Medium

EPA Acceptable

High

Extreme

0.00 mg Ni/L

0.05 mg Ni/L

0.02mg Ni/L

0.70mg Ni/L

1.10 mg Ni/L 1.60

mg Ni/L

3.00mg Ni/L

Hypothesis Affirmed

• Increased respiratory functions at 0.20 mg/L concentration of nickel solution

• Trend indicated that as nickel concentrations increased, biofilm respiration decreased

Future Work

• Repeat the experiment:

• Add more replicates per sample

• Use nickel as the cation for 2 solutions

• Nickel Chloride

• Nickel Nitrate

Acknowledgments

• Dr. Sally Entrekin: Assistant Professor of Biology at the University of Central Arkansas (UCA)

• Dr. Robert Mauldin: Chair of Chemistry Department at UCA

• Dr. Patrick Desrochers: Professor of Chemistry at UCA

• Adam Musto: Graduate Student in the Biology Department at UCA

• Department of Biology at UCA• Department of Chemistry at UCA