Nonoxidizing Biocides,

Thermal Shocking,

Desiccation, Oxygen

Deprivation, Carbon Dioxide

and Low Frequency Agitation

Robert F. McMahon

Department of Biology

The University of Texas at Arlington

Arlington, Texas 76019

Nonoxidizing Biocides,

Thermal Shocking,

Desiccation, Oxygen

Deprivation, Carbon Dioxide

and Low Frequency Agitation

Robert F. McMahon

Department of Biology

The University of Texas at Arlington

Arlington, Texas 76019

What is a Nonoxidizing Molluscicide?

• A molluscicide which does not act as a chemical oxidant• Does not induce corrosion of metallic surfaces• Organic or metallic molluscicides• Typical oxidizing molluscicides

• Chlorine, Sodium Hypochlorite• Chlorine dioxide• Chloramine• Bromine• Bromine, Bromine/Chlorine • Ozone• Potassium Permanganate, • Hydrogen peroxide

COMMERCIAL NONOXIDIZING MOLLUSCICIDES

Poly[oxyethlene(dimethyliminio)ethylene (dimethylimino)ethylene dichloride]Bulab 6002 (Buckman Laboratories)0.5 ppm for 826 h - 100% Adult Mortality2 ppm for 313 h - 100% Adult Mortality8 ppm for 197 h - 100% Adult Mortality

2-(Thiocyanomethythio)benzothiazoleBulab 6002 (Buckman Laboratories)0.5 ppm for 758 h - 100% Adult Mortality2 ppm for 313 h - 100% Adult Mortality4 ppm for 260 h - 100% Adult Mortality

Didecyl dimethyl ammonium chlorideH-130 (Calgon)1 ppm for 24 h - 100% Adult Mortality

N-alkyl dimethyl benzyl ammonium chloride &Dodecylguanidine hydrochlorideClam-trol - 1 or CT-1 (Betz Industrial)15 ppm for 12 h at 11°C - 100% Adult Mortality after 48 h15 ppm for 14 h at 14°C - 100% Adult Mortality after 48 h15 ppm for 6 h at 20°C - 100% Adult Mortality after 24 h15 ppm for 14 h at 20°C - 100% Adult Mortality after 48 h

N-alkyl dimethyl benzyl ammonium chlorideClam-trol-2 or CT-2 (Betz Industrial)2-5 ppm applied for 6-24 h - 100% Adult Mortality

N-alkyl dimethyl benzyl ammonium chlorideClam-trol - 4 or CT-4 (Betz Industrial) 13 ppm for 72 h at 5° or 10°C - >90% Adult Mortality13 ppm for 48 h at 15°C - 100% Adult Mortality13 ppm for 12 h at 20°C - 90% Adult Mortality

Akyldimethylbenzyl ammonium chloride & Akyldimethylethylbenzyl ammonium chlorideMactrol 9210 (Nalco)0.5 ppm for 249 h at 18°C - 100% Adult Mortality0.5 ppm for 120 h at 22°C - 100% Adult Mortality2.0 ppm for 65 h at 18°C - 100% Adult Mortality2.0 ppm for 45 h at 22°C - 100% Adult Mortality

Compound with Primary and Secondary Aminated Carbon ChainsMexel 432 (Mexel)2 ppm for 1.5h/day for 30 days - 40% Adult Mortality 10 ppm for 1.5 h/day for 30 days - 62-77% Adult Mortality

Dichloro-2'nitro-4' salicylanilideBayluscide (Bayer)0.05 ppm for 24 h - 70% Adult Mortality0.1 ppm for 24 h - 100% Adult Mortality

N-triphenylmethyl-morpholineFrescon (Shell)0.5 ppm for 24 h - 70% Adult Mortality0.9 ppm for 24 h - 100% Adult Mortality

Benzalkonium chloride (Fish Culture Disinfectant)10 ppm for 20 min - 43% Veliger Mortality after 24 h100 ppm for 20 min - 80% Veliger Mortality after 24 h1000 ppm for 20 min - 100% Veliger Mortality after 24 h

Tert-butyhydroxyquinone in paintsTBHQCan reduce but not eliminate settlement

Butylated Hydroxytoluene in paintsBHTCan reduce but not eliminate settlement

Surfactant AgentTD-2335 (Elf Atochem North America)1-1.5 ppm for 6-8 h - 100% Adult Mortality48 hr LC50 = 0.48 ppm (juveniles), 0.59 ppm (adults)

1,1'-(Methyliminio)bis(3-chloro-2-propanol), polymer with N,N, N',N'-tetramethyl-1,2-ethanediamineBulab 5001 (Buckman Laboratories)3 ppm for 1295 h at 20°C - 100% Juvenile Mortality9 ppm for 346 h at 20°C - 100% Juvenile Mortality3 ppm for 1295 h at 20°C - 100% Adult Mortality9 ppm for 633 h at 20°C - 100% Adult Mortality

Petroleum Jelly, Lanolin, Zinc oxide, Talc, Petroleum distillates (Major ingredients), Panthenol, Sorbitan sesquioleate, cetylridinium chloride, Allantoin, Witch Hazel (Minor ingredients)Penaten-Creme (Johnson and Johnson Co.)Diaper ointment used in Europe as an antifoulant compound. Inhibits adult mussel byssal re-attachment and pediveliger initial attachment

Aluminum sulfate (Alum)Al (SO )

Copper sulfate in SolutionCuSO100 ppm for 5 h at 22.5°C - 40% Adult Mortality300 ppm for 5 h at 22.5°C - 55% Adult Mortality

Tri-butyl tin oxideApplied in Surface Coatings Every 1-2 YearsInhibits Settlement (Not generally permitable)

ZincHot Metal Spray, in Paints or GalvanizationInhibits Settlement

CopperHot Metal Spray, in Paints or GalvanizationInhibits SettlementIons in solution (cathodic release)5 ppm for 24 h - 100% adult kill

Potassium ion in solutionAs 160-640 ppm KH PO - 100% Veliger MortalityAs 10 ppm KOH - 100% Veliger MortalityAs 50 ppm KCl - 100% Adult Mortality

NONCOMMERCIAL NONOXIDIZING MOLLUSCICIDESSaponin Compounds Extracted from the Berries of the African Soapberry PlantEndod15 ppm continuously applied - 100% Adult Mortality

Alkaloid Extract of Capsaicin Pepper PlantsCapsaicin

20.1±1.10 µM continuous - 90% inhibition of byssal attachment without mortality

N-vanillylnonanamide25.4±5.3 µM for 48h - 90% inhibition of byssal attachment without mortality

N-benzoylmonethanolamine benzoate 58.4±4.6 µM for 48 h - 90% inhibition of byssal attachment without mortality

(+)-Butanedioic acid, Mono[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6-yl]ester 50 ppt for 48h – Inhibits byssal attachment

(+-)-3,4-Dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-carboxylic acid50 ppt for 48h – Inhibits byssal attachment

(Poly-N-acetyl--D-glucosamine)-protein100 ppt for 48 h – 100% adult mortality

Marine Extracts of Macrophytic Algae (Fucus and Ulva) In coatings (Experimental)Inhibits pediveliger settlement and adult byssogenesis

Comparison of Nonoxidizing and Oxidizing Molluscicides

ADVANTAGES of NONOXIDIZING MOLLUSCICIDES

•Fewer precautions required for on-site storage

•Fewer safety hazards in handling and use

•May have greater toxicity than oxidizing agents

•Do not generally induce valve closure

•Surfactant agents may be less toxic to nontarget organisms than oxidizing molluscicides

• Due extensive surface area of living epithelial tissue in bivalves

•Application technology and hardware is often inexpensive and readily installed

•Readily and simply deactivated in effluents

•Generally noncorrosive to metal, silicone and rubber based seals

•Do not produce carcinogenic by-products (THMs)

Nonoxidizing Molluscicide Application Strategies

• Periodic Mitigation

• Continuous Application

• Intermittent Application

• Semicontinuous Application

Periodic Application

Short-term molluscicide addition at elevated concentrations mitigating established populations •Applied at a frequency preventing system degradation (1-3 times annually)

•Adjusted to local population reproductive cycles

Advantages:•Low annual cost and molluscicide usage

Disadvantages:•Requires extensive and continuous monitoring•Allows mussel fouling to become established•May release shells and bodies into system•High application levels may require discharge detoxification

Continuous Application

Continuous application at low concentrations•Prevent pediveliger settlement•Prevent survival of translocating juvenile musselsAdvantages:•Prevents establishment of mussel fouling•Application technology is relatively simple•Discharge molluscicide concentrations are low, but continuous•Reduces microbially influenced corrosion (MIC)•Extensive mussel monitoring is not requiredDisadvantages:•Relatively high annual use of molluscicide•Relatively high costs•High annual release of molluscicide

Intermittent Application

Daily, bidaily, or tridaily at intermediate concentrations for 0.5 to 3 hours per addition•Prevents pediveliger settlementAdvantages:•Slows establishment of mussel fouling•Discharge of molluscicide reduced compared to continuous application•Reduces costs•Reduces microbially influenced corrosion (MIC)•Can be limited to mussel spawning periods

Disadvantages:•Cannot mitigate pre-established fouling •Will not generally prevent fouling by translocating juvenile and adult mussels•Must monitor juvenile and adult settlement

Semi-continuous ApplicationRapid, on-off cycling of molluscicide addition at low concentrations as used in continuous application •On 15-30 min followed by off periods of 15-90 min

• Applied at low concentrations as in continuous application

Advantages:•Prevents establishment of mussel fouling•Discharge of molluscicide reduced compared to continuous application, reducing costs•Reduces microbially influenced corrosion (MIC)•Will mitigate pre-established mussel fouling

Disadvantages:

•Involves larger annual use of molluscicide than in periodic or intermittent application

•Application technology may be complex

Conclusions

• Nonoxidizing molluscicides offer a viable alternative to oxidizing molluscicides for zebra mussel fouling

• Wide variety of nonoxidizing molluscicides available

• Are highly effective against zebra mussels and some are relatively benign to nontarget species compared to oxidizing molluscides

• Application technology and storage are simple

• Costs can be competitive with oxidizing biocides

• Can reduce microbially influenced corrosion (MIC)

• Readily applied to low volume facilities and systems

• Choice of agent and application technology can be customized to facility requirements and limitations on a site-by-site basis

Thermal Control Strategies

• Zebra mussels are northern temperate species

• Results in having reduced tolerance of elevated temperatures

• Can tolerate 0°C throughout the winter

• Do not tolerate prolonged exposure to 30°C• Incipient upper thermal limit

• Low upper lethal limits make zebra mussels susceptible to thermal mitigation/control technologies

• Two basic thermal treatment technologies• Chronic thermal treatment• Acute thermal treatment

ACUTE THERMAL TOLERANCE

• Tolerated temperature in response to rapidly increasing temperature or instantaneous exposure to a lethal temperature

• Affected by of rate of temperature increase and prior temperature experience

CHRONIC THERMAL TOLERANCE

• Tolerance time when continuously exposed to lethal temperatures

• Affected by exposure temperature and prior temperature experience

Tem

pe

ratu

re

Tem

pe

ratu

re

Time Time

ACUTE versus CHRONIC THERMAL TREATMENT

Acute Treatment Chronic Treatment

Upper Lethal Threshold

Upper Lethal Threshold

Acute Temperature Tolerance

0.00 0.20 0.40 0.60 0.80 1.00

Temperature Increase Rate (°C/min)

33

34

35

36

37

38

39

40

41

0°C

5°C

10°C

15°C

20°C

25°C

30°C

AcclimationTemperature

Tole

rate

d T

emp

erat

ure

(L

T50

in °

C)

ApplicationsSteam or hot water injection

> 38°C (100°F) produces near instantaneous death under all conditions

EmbaymentsCompletely heatDewatering technique

Injection into piping

Steam or Hot Water Injection Raising Embayment Surface Water to a Lethal Temperature

Dewater Embayment Exposing all of Walls to Hot Water

Acute Thermal Treatment of Embayments

Circulating Water System

Service Water System

Valve

Inta

ke

Str

uc

ture

SourceWater

Intake

Discharge

Re-circulation of Thermal Effluents

CHRONIC THERMAL TREATMENT

Chronic Temperature Tolerance

31 32 33 34 35 36 37

Treatment Temperature (°C)

0

50

100

150

200

250

0°C

5°C

10°C

15°C

20°C

25°C

30°C

AcclimationTemperature

Su

rviv

al T

ime

(h)

Applications

Re-circulation of discharge water into intakes to raise and hold system temperatures at lethal levels (> 33°C, 91°F) for long enough to mitigate zebra mussel infestations

Ambient Water Temperature at Collection (°C)

10

20

30

40

50

60

70

0 5 10 15 20 25 300

15°C Acclimated

25°C Acclimated

5°C Acclimated

Mississippi River at Baton Rouge, LA

Mea

n S

urv

iva

l Tim

e at

33°

C (

Ho

urs

)Seasonal Thermal Acclimatization of Zebra Mussels will

also Affect Choice of Temperature and Application Times in Chronic Thermal Treatments

DesiccationZebra mussels are highly intolerant of emersion and desiccation

Makes dewatering of mussel infested structures a efficacious alternative to chemical mitigation

Three available strategiesAmbient air exposure, forced warmed air, or freezing

At ambient temperaturesIncreased kill rates at

higher temperaturesDecreased kill rates at

higher R.H.

Rapid kill in air warmed above lethal temperature

0 5 10 15 20 25 30

TEMPERATURE (°C)

0

50

100

150

200

250

300

350

400

LT50

100%80%

60%

40%20%0%

0 5 10 15 20 25 30

TEMPERATURE (°C)

0

100

200

300

400

500

600

700

800

LT100

80%60%

40%

0%

100%

20%

Ho

urs

Su

rviv

ed

Ho

urs

Su

rviv

ed

Desiccation tolerance of zebra mussels at different ambient air temperatures and relative humidities

< 5% 33% 53% 75% > 95%Relative Humidity

0

10

20

30

40

5035°C

LTLT

100

50

100% Sample Mortality

Ho

urs

Su

rviv

edSurvival of Zebra Mussels after Emersion in

a Lethal Air Temperature of 35°C

Note that survival is reduced in higher relative humidities

FreezingZebra mussels are highly intolerant of emersion in subfreezing temperatures

Could mitigate mussel infestations by dewatering during freezing conditions

Milton Matthews

-10 -7.5 -5 -3.0 -1.5 0.0

Air Temperature (°C)

0

5

10

15

20

25

30> 48 h > 48 h

LT 50 - Separate

SM 100 - Separate

LT 50 - Clustered

SM 100 - Clustered

Ho

urs

Su

rviv

ed

Tolerance of Aerial Freezing in Separate and Clustered Zebra Mussels

Field Test of Freeze Treatment

Black Rock Lock, Buffalo, New York, dewatered over a 6 hour period during January 1995

Infesting mussels were alive on all levels of the lock walls after dewatering when air temperatures were > 0°C

Air temperatures fell to -5°C over night

100% mortality of exposed mussels the following morning

Lock mussel infestation was completely mitigated

Oxygen Deprivation

Zebra mussels have very poor tolerance of hypoxia compared to other freshwater bivalves

Survival times are shorter and minimal tolerated levels of hypoxia are greater

Survial times decrease with decreasing oxygen concentration and increasing temperature

Could be used for nonchemical mitigation/control of zebra mussel fouling

Methods: Anoxia / Hypoxia Tolerance

Mussels acclimated to 5°, 15° or 25°C for > 14 daysRespiratory responses to progressive

hypoxia determined at 5°, 15° and 25°CTolerance of 0, 5, 10% of air oxygen

saturation tested Po = 0.0, 7.9 and 15.9 Torr

Responses of both D. polymorpha (zebra mussels) and D. bugensis (quagga mussel) tested

2

50%

60%

70%

80%90%

Oxygen Concentration

Oxy

gen

Up

take

Rat

e

Analysis of Oxygen Uptake Rates in Response to Progressive Hypoxia using

% Oxygen Regulation Values

Full air oxygen saturation (159 Torr)

5 15 20 250

20

40

60

80

100 Dreissena bugensis

Dreissena polymorpha

Acclimation / Test Temperature (°C)

Per

cen

t O

R

egu

lati

on

2

Species Condition Temp. LT50 Log RankD. bugensis 5%O2 15°C 19 Days A D. polymorpha 5%O2 15°C 24 Days BD. bugensis 5%O2 25°C 6 Days AD. polymorpha 5%O2 25°C 5 Days AD. bugensis 10%O2 15°C 26 Days A D. polymorpha 10%O2 15°C 28%@ 30 Days BD. bugensis 10%O2 25°C 6 Days AD. polymorpha 10%O2 25°C 15 Days BD. bugensis Anoxia 15°C 18 Days AD. polymorpha Anoxia 15°C 32 Days BD. bugensis Anoxia 25°C 5 Days AD. polymorpha Anoxia 25°C 16 Days B

LT50 values (days) (Kaplan-Meier Survival Analysis) and Log Rank Statistic comparing the hypoxia (5% air O2 saturation) and anoxia tolerances of specimens of D. bugensis and D. polymorpha.

Carbon Dioxide Treatment

• Carbon dioxide is a weak acid• CO2 + H2O → H2CO3 → H+ + HCO3- → 2 H+ + CO3

2-

• Bivalves do not have pH buffering blood proteins

• Increased CO2 concentration in medium increases CO2 concentration in blood lowering its pH

• Slight reductions in blood pH induce stress and mortality so that CO2 could act as a molluscicide

• Inexpensive, readily stored on site, easily applied, highly biodegradable

• Utilized by aquatic plants, algae and bacteria

Methods: Carbon Dioxide Tolerance

• Treatment temperature = 25°C

• Mussels exposed to 5% and 10% CO2

• Pco2 = 38 and 76 Torr, respectively

• Tolerance times determined

• Mussels allowed to byssally attach to clear plastic plates over the course of the exposures

• Number of new byssal attachment produced determined daily

0 4 8 12 16 20

0

20

40

60

80

100LT = 2.59 Days

LT = 4.73 Days

LT = 5.63 Days

Days of Exposure

Cu

mu

lati

ve %

Mo

rtal

ity

Mortality in 10% CO / 19% O / 71% N2 2 2

5050

50

0 1 2 3 4 5 6 7 8 9 10 110

2

4

6

8

10

12 Air

Days

The Effects of Exposure to 5% CO on the Rate of Byssal Thread Production

2

5% CO 2

0 1 2 3 4 5 6 7 8 9 10 110

102030405060708090

100Air

5% CO2

Days

Pe

rce

nt

Att

ac

he

d

The Effects of Exposure to 5% CO on the Percent of Mussels Remaning Byssally Attached

2

Low Frequency Agitation

• Low frequency agitation (20-60 cps) can inhibit production of byssal threads and induce byssal release in zebra mussels

• May be the reason that adult mussels are not found in shallow waters agitated by wave action

• Could be used as a nonchemical method for controlling or mitigating fouling• Low frequency agitators in embayments• Flutter valves in water lines or intake tunnels• Bubble Sreens, Bubbler Systems

• Higher frequency sound has not proven successful

Methods: Low Frequency Agitation

• Mussels allowed to attach to plastic plates• Water agitated back and forth past the plates

• 0, 10, 20, 30, and 40 cycles/min (cpm)

• Number of byssal threads produced counted daily

• Number of mussels sontaneously releasing from byssus observed

0 2 4 6 8 10 12 14 16 18 20

20

40

60

80

100

Days

Me

an

By

ss

al T

hre

ad

Nu

mb

er

40 CPM

15 CPM

0 CPM

30 CPM

10 CPM

Effect of Low Frequency Agitation on Cumulative Byssal Thread Production

Agitation Rate (CPM)

Me

an

Bys

sal

Th

rea

d N

um

be

r

0 10 20 30 40

40

60

80

100

120

Effect of Low-frequency Agitation on Mean Number of Byssal Threads Produced after 21 Days

Above 40 cpm all mussels spontaneously released from the plastic plate

Use of Bubble Screens and Plates for Mitigation of Zebra Mussel Macrofouling

Conclusions

• Thermal treatments successfully mitigate and control dreissenid macrofouling

• Acute and chronic treatments

• Anoxia/hypoxia could be efficacious for mitigation of zebra mussel infestations

• Best applied during warmer months• Will require reducing oxygen concentrations to less than 15% of full

air oxygen staturation• Drawing from deoxygenated hypolimnetic waters

• Desiccation has been successfully used to control mussel macrofouling

• Draw-down during warm summer or freezing winter periods• Dewatering components• Forced warm air

• Carbon dioxide injection could an efficacious mitigant• 10% will kill mussels, 5% stimulates release from byssus

• Low frequency agitation could be an effective mitigant• > 50 cpm causes 100% byssal release• < 40 cpm inhibits production of byssal threads• Flutter valves, agitators or bubble screens could prevent settlement