Sample_Contract.pdf

Sam ple

STATE OF CALIFORNIA - DEPARTMENT OF GENERAL SERVICES

STANDARD AGREEMENT

STD 213 (Rev. 04/2020)

AGREEMENT NUMBER

D2380005 PURCHASING AUTHORITY NUMBER (If Applicable)

SCO ID: 3600-D2380005(SAMPLE)

1. This Agreement is entered into between the Contracting Agency and the Contractor named below:

CONTRACTING AGENCY NAME

California Department of Fish and Wildlife

CONTRACTOR NAME

To Be Determined (TBD)

2. The term of this Agreement is:

START DATE

Upon California Department of Fish and Wildlife Approval

THROUGH END DATE

March 31, 2026

3. The maximum amount of this Agreement is:

TBD

4. The parties agree to comply with the terms and conditions of the following exhibits, which are by this reference made a part of the Agreement.

Exhibits Title Pages

Exhibit A Scope of Work 5

Exhibit A, Attachment

Husbandry Manual for Rana sierrae at the San Francisco Zoo, July 2017 30

Exhibit B Budget Detail and Payment Provisions 4

Exhibit C * General Terms and Conditions (GTC 04/2017) *

Exhibit D Additional Provisions 10

Items shownwith anasterisk (*), are hereby incorporated by reference andmadepart of this agreement as if attachedhereto.

These documents can be viewed at https://www.dgs.ca.gov/OLS/Resources

INWITNESSWHEREOF, THIS AGREEMENTHAS BEENEXECUTEDBY THE PARTIES HERETO.

CONTRACTOR

CONTRACTOR NAME (if other than an individual, state whether a corporation, partnership, etc.)

TBD

CONTRACTOR BUSINESS ADDRESS

TBD

CITY

TBD

STATE

TBD

ZIP

TBD

PRINTED NAME OF PERSON SIGNING

TBD

TITLE

CONTRACTOR AUTHORIZED SIGNATURE DATE SIGNED

STATE OF CALIFORNIA - DEPARTMENT OF GENERAL SERVICES

STANDARD AGREEMENT

STD 213 (Rev. 04/2020)

AGREEMENT NUMBER

D2380005 PURCHASING AUTHORITY NUMBER (If Applicable)

SCO ID: 3600-D2380005(SAMPLE)

STATE OF CALIFORNIA

CONTRACTING AGENCY NAME

California Department of Fish and Wildlife

CONTRACTING AGENCY ADDRESS

P.O. Box 944209

CITY

Sacramento

STATE

CA

ZIP

94244

PRINTED NAME OF PERSON SIGNING

Amy Mowrer

TITLE

Branch Chief, Business Management Branch

CONTRACTING AGENCY AUTHORIZED SIGNATURE DATE SIGNED

CALIFORNIA DEPARTMENT OF GENERAL SERVICES APPROVAL EXEMPTION (If Applicable) Governor's Executive Order N-10-21

California Department of Fish and Wildlife Agreement Number: D2380005 Exhibit A – Scope of Work (Drought) Contractor: To Be Determined (TBD)

(Version 03/2023)

1. Introduction of Services

The Contractor agrees to provide, to the California Department of Fish and Wildlife (CDFW), wildlife care and facilities to rescue and maintain populations via captive rearing of frogs that are California Endangered Species Act (CESA) and/or Federal Endangered Species Act (ESA) listed as threatened or endangered, in accordance with the specifications, terms, and conditions contained herein.

2. Location of Services

The services will be performed at the Contractor’s place of business:

TBD

3. Service Schedule

The services will be provided between normal business hours, except on holidays. The Contractor will be required to obtain agreement, in writing, from the CDFW Contract Manager for any changes to the service schedule.

4. Project Officials

CDFW Project Officials Contractor Project Officials

Contract Manager Project Director Name: Ryan Peek Name: TBD Phone: (916) 838-1611 Phone: TBD Email: Ryan.peek@wildlife.ca.gov Email: TBD Address: P.O. Box 944209

Sacramento, CA 94244-2090 Address: TBD

Direct all inquiries to: Direct all inquiries to:

CDFW – Wildlife Branch Name: Joshua Thornton Name: Same as above Address: P.O. Box 944209 Address:

Sacramento, CA 94244-2090 Phone: (916) 530-0843 Phone:

Email: Joshua.thornton@wildlife.ca.gov Email:

Either party may make changes to the Project Officials by giving written notice to the other party. Said changes will not require an amendment to this Agreement.

5. Scope of Work

A. BACKGROUND AND OBJECTIVES

mailto:Ryan.peek@wildlife.ca.gov mailto:Joshua.thornton@wildlife.ca.gov

The Governor’s Executive Order N-10-21 directs the CDFW to immediately implement projects that respond to drought conditions through habitat restoration and through water infrastructure projects on property owned or managed by CDFW for the benefit of fish and wildlife impacted by the drought. The Executive Order authorizes CDFW to enter into contracts to accomplish this work and suspends provisions of the Government Code, Public Contract Code and California Environmental Quality Act relative to implementation of these habitat restoration and water infrastructure projects.

The compounding effects of long-term drought and climate change are resulting in hydrologic changes to habitats on which native amphibians rely. Drought conditions can directly impact native frog species via reductions in the water level, loss of surface water earlier in the water year, and poorer water quality. Less water availability and more rapid drying of aquatic habitats is especially detrimental in breeding and rearing locations where desiccation or isolation of larvae can increase the risk of mortality. Therefore, collecting early life stages (e.g., eggs, larvae, or recent young of year) is necessary to prevent complete loss of cohorts among populations of concern. Captive rearing provides one of the best options currently available to increase the odds of survival for larval frogs that require intervention in the event of stranding and habitat desiccation. Captive rearing involves collecting early life stages, bringing individuals into an accredited conservation facility, raising the frogs to adulthood under conditions that mimic those in the wild, and then releasing the mature individuals back into their native habitat.

Funding will provide the Contractor with funds to raise up to 200 individuals/cohort of native frogs per year. The Contractor must have expertise in the care, maintenance, medical related care/procedures, and breeding of target species, and remain a close partner with CDFW and other agencies leading conservation efforts for native frogs. The Contractor will work closely with CDFW throughout the entire process, from collection to release, following the aforementioned captive rearing protocols, Institutional Animal Care and Use Committee (IACUC) requirements, and policies described in the Interagency Conservation Strategy for Mountain Yellow-legged Frogs in the Sierra Nevada (MYLF ITT 2018, Attachment 5).1

B. WORK TO BE PERFORMED

The Contractor will provide CDFW with the following:

Task 1: Captive-rearing native amphibians to maturity

This will be the Contractor’s primary task. The Contractor will receive early life stage frogs from partner biologists including CDFW, United States Forest Service (USFS), U.S. Fish and Wildlife Service, National Park Service (NPS), U.S. Geological Survey (USGS), environmental consultants, and university research staff. Following delivery, the Contractor will raise early life stage amphibians to maturity following the captive-rearing procedures and protocols outlined in Exhibit A, Attachment 1 - Husbandry

1 fws.gov/sites/default/files/documents/Mountain-Yellow-Legged-Frog-Conservation-Strategy.pdf https://www.fws.gov/sites/default/files/documents/Mountain-Yellow-Legged-Frog-Conservation-Strategy.pdf https://www.fws.gov/sites/default/files/documents/Mountain-Yellow-Legged-Frog-Conservation-Strategy.pdf https://www.fws.gov/sites/default/files/documents/Mountain-Yellow-Legged-Frog-Conservation-Strategy.pdf https://www.fws.gov/sites/default/files/documents/Mountain-Yellow-Legged-Frog-Conservation-Strategy.pdf

Manual for Rana sierrae at the San Francisco Zoo, July 2017. A summary of the manual is outlined below.

• The Contractor will maintain the animal care staff, who will provide the daily maintenance of tadpoles and frogs (includes feeding, cleaning, weighing and other related care).

• The Contractor will test metamorphosed frogs for amphibian chytrid (Batrachochytrium dendrobatidis; Bd) and treat accordingly with antifungal medication.

• The Contractor will provide access to lab-work/testing as needed or required for pre-release health screening and regular health care checks.

• The Contractor will provide pathology findings for deceased specimens for disease monitoring.

• The Contractor will provide quarantine facilities appropriate for frogs and with appropriate protocols and policies to ensure best care practices and welfare of live animals (filtered tanks and other structures appropriate for the species in question).

• The Contractor will provide infrastructure to support care of frogs including but not limited to access to water, plumbing, electricity, secure animal care areas, veterinary labs, and animal care kitchens.

• The Contractor will maintain animal records utilizing the Zoological Information Management Software (ZIMS) Species 360 database for husbandry and medical records.

• The Contractor will provide summary reports to CDFW quarterly and annually.

• The Contractor will notify CDFW (contacts being the same as in “REPORTS” below) within 72 hours in the event of significant unforeseen circumstances or medical events of concern among captive frog cohorts (e.g., sudden mortality events, disease outbreaks, observations of above average malformation rates, etc.) and discuss mitigations, troubleshooting, and next steps.

• The Contractor will transfer frogs under the guidance and authority of CDFW, either to rewild once suitable and appropriate conditions exist, or for other appropriate procedures as deemed necessary by CDFW (for example, relocation).

Task 2: Prepare captive-reared amphibians for release back into the wild

Once amphibians have been raised to maturity, the Contractor staff will prepare individuals for release back into the wild. The Contractor will coordinate with CDFW and other agency partners on the timing of pickup, delivery, and release back into the wild.

Details on all of these processes are provided below:

• All mature individuals ready for release will receive an 8 x 1.4 mm passive integrated transponder (PIT) tag inserted beneath the dorsal skin to provide a permanent, unique identifying marker to each frog.

• Prior to release, a subset of frogs from each frog-housing tank will be tested for Bd infection. If Bd is detected on any of the frogs, all individuals from the tank(s) with positive Bd detection(s) will be treated with antifungal drugs to clear the infection.

Subsequent clearance of infection will be confirmed via additional skin swab analysis following administration of antifungal medication.

• In the week prior to planned release, the Contractor staff will produce a list including the PIT tag number, sex, snout-to-urostyle length, and weight of each frog.

• On the day of collection for release, all frogs will be housed individually inside clean 4–8 oz. containers that contain air holes and wetted filter paper. Each container will include a label that indicates the frog’s sex.

• On the day of release, the Contractor staff will provide a signed chain-of-custody form to the authorized individuals responsible for receiving the frogs and delivering the frogs back into the wild.

C. REPORTS

For any years during which any cohort(s) of early life stage frogs are brought to the Contractor for captive rearing funded by this contract, Contractor will supply a summary report via email to CDFW contacts, including the Statewide Amphibian and Reptile Conservation Coordinator (Laura.Patterson@wildlife.ca.gov), CDFW Drought Contract Manager (Ryan.Peek@wildlife.ca.gov, or a CDFW-designated alternate), and the local environmental scientist contact from the CDFW Region where frogs were collected.

Quarterly and annual reports will contain summary information on the species, numbers of individual frogs received, life-stage(s), sex (if known), and year-to-date totals. The report will include a Chain of Custody form for any cohorts collected and brought into the Contractor. The report will also contain descriptions of any significant event(s) or finding(s), including any experiments or research performed, and necropsy reports, when available. The Contractor will provide additional reporting on an as-needed basis, in the event of unforeseen circumstances/emergencies including but not limited to sudden mortality events, disease outbreaks, observations of above average malformation rates, and to discuss mitigations, troubleshooting, and next steps with

CDFW.

The Contractor will prepare and submit a draft annual report to the CDFW Contract Manager in Microsoft Word format by December 1 of each year of the contract.

The CDFW Contract Manager will have 30 calendar days to provide comments to the draft annual report, after which a final report must be prepared. The Contractor will produce a final annual report no later than March 1st (of the year following when the cohort was received) in Microsoft Word or PDF format.

Any report deadlines may be adjusted if CDFW agrees to either 1) delayed reporting, or

2) foregoing the need for any particular quarterly report. All changes to deadlines and scheduling must be agreed upon in writing by the Contractor and CDFW Contract Manager.

The CDFW Contract Manager may accept the draft report as the final report. The report will not be considered final until accepted and approved by the CDFW Contract Manager.

mailto:Laura.Patterson@wildlife.ca.gov mailto:Ryan.Peek@wildlife.ca.gov

D. Schedule of Completion Dates

Task Date

Collection and transport Late spring–early fall, annually (2024 and 2025) Completed by 10/15/2024; 10/15/2025.

Captive-rearing late summer 2024–early summer 2025, and late summer 2025– early spring 2026) Completed by 7/15/2025; 3/20/2026

Quarterly reports due First Monday of each Quarter

Draft annual reports due 12/1/2024; 12/1/2025

Final Project report March 31, 2026

6. Contractor Minimum Qualification/Requirements

The Contractor must have a history of rearing, releasing, and triaging local California herpetofauna. The Contractor must work closely to ensure timely and successful care for individual frogs. Frogs may be reared in collaboration within the Association of Zoos & Aquariums (AZA) community and field partners with shared husbandry resources and protocols.

The lead Contractor must have a post-graduate education in wildlife, husbandry, ecology or related field, and must have extensive experience managing wildlife conservation facilities, including conducting captive rearing of native amphibians.

7. Insurance Requirements

The Contractor will be required to maintain the following insurance coverage throughout the duration of this agreement and must supply proof of coverage upon request by CDFW.

Insurance must be in accordance with the Exhibit D CDFW Additional Provisions.

1. Commercial General Liability (limits not less than $1,000,000 per occurrence with a $2,000,000 annual policy aggregate).

2. Automobile Liability (limits not less than $1,000,000 combined single limit per accident).

3. Workers Compensation and Employers Liability (limits of $1,000,000 are required.

The waiver of subrogation endorsement must also be provided).

The above policies must each be endorsed to include “the State of California, its officers, agents and employees as additional insured but only with respect to work performed under the Contract”.

Husbandry Manual for Rana sierrae at the San Francisco Zoo

DRAFT July 2020

Taxonomic Classification

Kingdom Animalia

Phylum Chordata

Class Amphibia

Order Anura

Family Ranidae

Genus Rana

Species sierrae

Taxonomic Notes: Yellow-legged frog populations now recognized as Rana sierrae formerly were included in Rana muscosa. Vredenburg et al. (2007) examined phylogeography of Rana muscosa as defined by Stebbins (2003) and determined that R. muscosa occurs in the southern

Sierra Nevada and in mountains to the south and that populations in the Sierra Nevada north of this range comprise a distinct species (Rana sierrae). (IUCN website)

Conservation Status: Listed as Endangered in view of severe recent declines that likely are continuing (rate of decline over past 10 years unknown) and that has left only a small number of extant populations occupying a small fragmented area.

Zoo abbreviation for sex identification:

0 . 0 . 0

# of males

# of females

# of undetermined sex

Husbandry Considerations

Enclosures

As large an enclosure as possible should be used to provide maximum water capacity. A large water volume with proper filtration maintains more stable water quality parameters than a smaller water volume similarly equipped (a higher water volume can be achieved by use of a sump). A minimum volume of 20 gallons per enclosure must be used for both adult frogs and tadpoles.

Exhibit A, Attachment 1

All enclosures should ideally be constructed of easily disinfected materials like plastic, glass or fiberglass. In most cases as large an enclosure as possible should be used to achieve and maintain stable water quality within appropriate parameters. All containers should have screened or solid lids to prevent frogs from climbing or jumping out and escaping. An exception to this is the tadpole stage; however, once the larvae begin to metamorphose a lid should be placed on the aquaria or the metamorphosing tadpoles should be moved to a tank that has a lid.

Water depth should be 10-12 inches inches for swimming larvae and around 6 inches for metamorphosing larvae and froglets. Adult frogs are capable of thriving in deep waters and can be maintained at a water depth of 10 inches or greater.

Disturbances to the frogs should be minimized by setting up the holding containers in low human/animal activity areas or by covering the outsides of the aquaria with a visual barrier such as black shade cloth. If the frogs are to be released, they should be kept in strict quarantine holding as described in the AZA Amphibian Taxon Advisory Group’s Amphibian Husbandry

Resource Guide (Chapter 3; “Amphibian Quarantine Guidelines”). Additionally, yellow-legged frogs are very susceptible to chytrid infection and will not be housed outdoors to eliminate the risk of pathogenic contamination from native amphibians.

Equipment

Each enclosure should have its own set of designated equipment – nets, gravel siphon, water pumps, etc. and should not be shared with other enclosures.

Disposable vinyl or nitrile gloves should be used when handling metamorphosed frogs and should be changed between enclosures. This precaution should be taken to reduce the spread of any disease or contaminant from animal to animal and enclosure to enclosure. This is especially important when handling Bd+ animals recently brought in from the field or during immunization infections. Latex gloves must be avoided as they have been shown to have negative health effects in larval amphibians. Please do not use latex gloves in any of the frog rooms at any time.

All use of disposable gloves will be avoided when handling tadpoles and they should be handled only with bare hands.

Cage furniture and Plants

Hiding spots, basking spots and aquatic perches are essential for frogs to feel comfortable in their enclosures. Visual barriers are important to reduce stress between frogs within the same enclosure and to reduce stress caused by activity outside their enclosure. Examples include plastic plant pots that are placed on its side or upside down with a hole cut in the side large enough for the frog or froglets to enter and exit, sinking fish hides, and artificial floating plants that provide larvae and frogs with essential resting and hiding places.

Like plastic plants, live plants can provide a sense of cover and security for frogs and additionally aid in ammonia capture from the water. Before live plants are used in an enclosure they should be removed from their container and the roots should be rinsed in clean freshwater to remove soil. Following the freshwater rinse plants should be soaked in a dilute bleach solution

(0.4% sodium hypochlorite) dip for 10–15 minutes. Various plants will react differently, and some will not survive the treatment. For example, sturdy aroids like Pothos (Epipremnum spp.)

and Philodendron cordatum will fare better than will begonias, ferns, and other plants with delicate stems or leaves.

After the bleach treatment rinse plants overall to remove dirt or surface contaminants on stems and leaves. Plants should then be held at a temperature above 90 degrees for at least 96 hours to reduce the risk of introducing chytrid fungus. If they survive these treatments, the plants may then be transferred to new, sterile soil and containers or “planted” in containers with rocks and submerged in water. Many plants may be rooted and grown hydroponically (in water) which is a preferable method where applicable.

Platforms constructed of plastics such as PVC can be used to allow frogs to move higher up in the water column for thermoregulatory purposes. If tadpoles are housed in a glass aquarium then suction cups can be used to attach such platforms to the side of the tank. Floating turtle docks also work well for feeding platforms and haul out sites (ZooMed Turtle Docks TD-5 (mini), TD-

10 (small) and TD -20 (medium)) and are an ideal option for both tadpole and frog tanks. Each tank should have one or two docks of appropriate size to allow frogs to emerge for basking and for metamorphing tadpoles to have a way out of deep water.

Substrate

Traditionally, tadpole tanks have lacked substrate to allow for an ease of access to sinking food.

This is important when the tadpoles are very small and can easily penetrate between the rocks.

However, substrate, if installed correctly, can provide essential filtration for this life stage and additionally provide nutritional algae supplementation to their diet.

Frog enclosures should be composed of a substrate of large smooth rocks as well as large river rocks which can be used for feeding platform as well as provide suitable basking sites.

Two groups of rock sizes are well suited for substrate for frog tanks. Small pebbles approximately ½ cm in diameter are best for the area of the enclosure which is submerged in water. This small rock size allows for easy siphoning during cleaning. The land portion of the tank (if there is one) should have large rocks at least 4 cm in diameter. This large rock size is required to prevent frogs from swallowing rocks while in pursuit of prey items. Rocks smaller than 4 cm can become lodged in the stomach causing the digestive system to shut down.

Removal of rocks from the gut of a frog is a difficult and risky process so selecting the right substrate is essential. Generally, the SF Zoo uses only larger rocks throughout the tanks for ease of consistency.

Lighting & Temperature

Each frog or tadpole tank should contain a digital probe thermometer for daily water temperature monitoring. Each separate room should also contain a thermometer capable of recording daily high/low and current temperature. Temperature is one of the most important factors in housing this species. Care must be taken to insure that their housing temperatures are within natural range. According to experts, temperatures over 80°F could be deadly to this animal.

An ambient temperature range of 10–18 °C (50–65 °F) should be offered, with a specific hot spot of 29-32°C (84 – 90°F). This will provide a thermal gradient that allows the frog to select desired temperatures for proper behavior thermoregulation.

Artificial lighting can be used to simulate natural sunlight at levels approximately equal to the wild habitat using compact fluorescent lights, set on an ambient timer, to simulate daylight in an indoor set up. Ambient lighting should consist of fluorescent bulbs in a four-foot fixture set on a timer. Each Fixture should contain 1 ZooMed 10.0 UVB bulb and 1 ZooMed Nature Sun bulb.

Ultraviolet B lighting should be provided by using either: A 100 watt flood light, Mega –Ray mercury vapour UVB lamp (Reptile UV Tel (252) 240-9088) or 125 watt Westeron Reptile Light with UVB. This will allow frogs to exhibit the basking behavior exhibited by wild frogs and provides a heat gradient in the tank. The photoperiod should roughly follow seasonal changes, adjusting time clock seasonally.

UVB basking sites are on a seasonal schedule:

Summer – 4 hours

Spring and Fall – 3 hours

Winter – 2 hour (only for release animals, not for accessioned population which keep 3 hours)

Date the bulbs when installed and replace annually or as needed. UVB bulbs should be replaced when the UVB production dips below 50 nm. UVB readings are measured at the first of the month.

Proper placement of lights should be taken into consideration. Multiple basking sites should be provided as haul out areas and as underwater perches for tadpoles. Light should also be provided in a patchwork mosaic so an animal can choose between light and dark spaces to provide light and dark refugia for tadpole security. Frogs should have ample haul out areas with UVB light to bask (as well as some without UV). Without multiple perching areas, smaller frogs will get outcompeted for access to the light/heat.

Water temperature should be maintained between 10 and 18 degrees Celsius (50 and 65 degrees

Fahrenheit) with seasonal changes to mimic natural temperatures. To accommodate this temperature fluctuation may require chillers to be attached to each tank. Ideally for resident adult population there should be some temperature variation; however, since our residents are not breeding or able to be released, the temperature fluctuations do not need to be significant.

Recommendations from San Diego Zoo were:

Fall 13°C (55.4°F)

Winter 10°C (50°F)

Spring 14°C (57.2°F)

Summer 18°C (64.4°F)

For the head-start group, mostly due to HVAC capacity but also to aid us to keep head-starts growing and eating regularly throughout the year, we keep head-start rooms at or around 60-

64°F (16 -18°C).

For breeding animals, adjust the water temperatures seasonally according to the following guidelines to safely provide adults a hibernation period that will be lowered to 4°C in the winter.

Use of a high quality chiller is essential for hibernation in their holding tank.

Early-Fall 13°C (55.4°F)

Late-Fall 10°C (50°F) and do not feed for 3-4 weeks

Winter 4.5°C (40°F) by slowly reducing temp 1°F every other day for one month

Spring 13°C (55.4°F) by slowly bring up temp1°F every other day for one month

Summer 18°C (64.4°F)

Life Stage Specific Considerations

Housing - Embryos

In general, egg mass enclosures should be large enough so that the pump produces minimal current to agitate the egg mass or recently hatched larvae. San Diego Zoo has held their egg masses at 13°C (55.4°F) and tadpoles should be held between 13°C and 16°C (55.4°F-61°F).

However, keeping our tadpoles and egg masses at 16-18°C (62-64°F) has been successful.

Gently aerate water in embryo holding tank with a sponge filter and aquarium pump or aquarium power head. If a sponge filter is not available than an air stone may be used. A sponge filter is preferred as it provides some biological filtration if it has properly aged. An air stone does not provide any filtration.

Generally, if egg masses have been brought out of the field for head-starting, they will hatch within 1-2 weeks of arrival. If there are some eggs that become fungus covered it likely will not affect water quality but monitor ammonia levels closely and do regular water changes. If captive breeding, we may need to remove dead hatchlings or eggs covered with fungus from the mass if possible, with minimal disturbance, using scissors and tweezers. As these eggs will be in the tanks for longer periods of time, ammonia levels can rise to a toxic level from decomposing eggs or hatchlings even with biological filtration.

Stocking density:

One egg mass (up to 1000 eggs) per 20 gallons of filtered aerated water.

Housing - Larvae

All use of disposable gloves will be avoided when handling tadpoles and they should be handled only with bare hands.

Mechanical, chemical and biological filtration is essential to maintain water quality. UV sterilizers should be also be used to reduce the density of aquatic bacteria and parasites.

External canister filters are best for maintaining high volumes of water and moderate to high stocking densities. Under gravel filters and sponge filters are best for low water volumes and low stocking densities.

Even with filtration, water changes are essential to reduce buildup of organic waste product.

Approximately 50 percent of the water volume should be changed weekly once the biological filtration is functioning; until then, daily water changes will be necessary to keep the ammonia level at acceptable amounts. Once established, we do 2-3 25% water changes per week based on water quality testing which is done daily.

Ideal temperature for water at tadpole stage is 55-64°F (13-18°C). With lower water temperatures the ammonia structure is less harmful than at warmer water temps (for further information see water quality document from AZA ATAG).

Systems that include algae growth and living plants are encouraged as it provides additional buffering of water quality parameters. Additionally, algae is excellent food for larvae.

Sizes can be mixed to a degree; with R. muscosa there is no evidence that large tadpoles harm small individuals. However, R. sierrae tadpoles will consume deceased tadpoles and there is some evidence that they may consume smaller tadpoles. Stocking capacity declines as tadpoles grow larger, so it is important to monitor water quality closely and check for signs of overcrowding.

For maximum growth:

25-30 larvae per 20 gallons of filtered, aerated water

Housing - Metamorphosing Larvae

Disposable vinyl or nitrile gloves should be used when handling metamorphosed frogs and should be changed between enclosures. Latex gloves will be avoided as they have been shown to have negative health effects in larval amphibians.

Closely monitor larvae showing developed hind limbs and the beginnings of forelimbs (Gosner stage 41). Water depth can be decreased to no more than 5 inches for this stage if there is a chance of complications or health concerns, or if it is difficult to find an individual metamorphing in the tank.

Edges of the enclosure should have haul out areas and underwater perches. Some larvae may drown swimming the perimeter of the enclosure looking for haul out areas if none have been provided. ZooMed Turtle Docks work well for haul out areas and also provide cover.

Some haul out areas should be beneath basking lights. Wattage of light should be adjusted to provide a hotspot of 29-32°C (84 – 90°F).

Newly metamorphosed froglets should be separated by size to keep cannibalism to a minimum.

Although larvae are not thought to be cannibalistic, juvenile and adult frogs are. Feeding frogs is also easier if separated into groups based on size.

Once tadpoles have reached Gosner stage 42 (forelimbs are fully emerged), they can be left in aquariums for up to 24 hours, especially if still foraging. However, after that period of time, they are removed and placed in a tanks with other metamorphosing froglets. If any health concerns exist or if there have been historic health issues with a population, the metamorphosing froglets are moved to a small, slanted tank with a dry area and 1-2 inches of water. This allows them to absorb their tails fully without fighting to swim and/or potentially drown. This is an issue often seen in the San Diego frogs. Tetney, a nuerological issue, has been seen in SDZ and SFZ during this life stage, is more easily identified and resolved in controlled smaller tanks. A ZooMed turtle block is put into the water to increase the hardness of the water. After about 7 days, the tail is nearly fully absorbed and food is introduced.

No more than 10 metamorphs or froglets per 10 gallon half tank enclosure.

Diet

Many of the problems with metamorphosis are due to poor nutrition as a tadpole. Mistakes during tadpole development may result in dying tadpoles, stunted metamorphs, or froglets that are unable to capture prey properly.

Rana tadpoles typically graze off the bottom of the water column or on the surface of objects.

Food should be placed on bottom of the water column to ensure the tadpole find it easily.

Type of food for larvae:

Processed fish foods:

Spirulina-based fish foods and algae wafers designed for herbivorous cichlids work well. Older tadpoles seem to especially prefer the algae gel (we use Repashy Superfoods Soilent Green meal replacement gel). Sinking wafers or pellets are preferred to floating wafers or pellets (such as

Hikari algae wafers).

Powdered variety can be mixed with a small amount of water and then poured onto shallow, non floating, plates that are then left to dry. Once dry these plates can be placed in the tadpole enclosures where they will graze off of them (we use Sera micron).

To keep water quality at a good level, tadpoles are offered food twice daily in small amounts.

They are also fed two different items to increase potential for them to eat. Current diet is:

Repashy gel in the morning – left for 2-3 hours

Sera micron plates in the afternoon and left overnight

Hikari algae wafers are occasional feedings or in place of the gel.

San Diego does not offer food ad libitum. Instead they feed for 24 hours then no food is offered for the next 24 hours to help keep water quality good.

Types of food for juveniles and adults

Frogs feed well on domestic crickets, mealworm larvae, mealworm adult beetles, flightless houseflies, wax worm larvae, and earthworms. Food must be offered alive except houseflies that are fed defrosted to tanks with a cycling water system which moves the flies around the enclosure.

Feeder crickets and mini-mealworms should be gut loaded with calcium. Additionally, insects should be dusted with calcium with D3 powder at every feeding to increase the calcium content ingested by the frog. Insects should also be dusted with a multivitamin powder every other feeding just prior to feeding out to the frogs.

Feeding frequency will vary with the simulated lab temperatures and conditions. During the spring and fall frogs should be offered live food two times per week, increasing to two-three times a week during the summer depending on appetite. During the winter juvenile frogs and non-breeding adults shall be offered food once per week when and if the water temperatures are brought down; otherwise, they are fed the same as the fall.

Any adult frogs which are hibernated during the winter shall be fasted for a period of two weeks prior to hibernation. During hibernation the frogs will not be fed.

Health and Water Considerations

Air Quality

If the air smells bad to you for any reason, it may contain chemicals that are harmful for amphibians. Avoid the use of strong smelling chemicals or solvents in the airspace around an enclosure. Make sure the ventilation leading to the amphibian enclosure does not communicate with any space that has dangerous chemicals in the air.

Water in enclosures should have sufficient aeration so that the larvae are not gasping for air at the top of the tank or looking distressed. This can be achieved by air stones, sponge filters, or allowing water from filter systems to splash in the water to increase aeration.

Water Quality and Changing Schedule

Water quality parameters for Rana sierrae in the wild are typical of granitic basins throughout the central and southern Sierra Nevada in having near-neutral pH and very low conductivity.

Therefore, the ph range of 7.0-7.3 (examples from Desolation) are simulated by adding 2 teaspoon of Kent Marine R/O Right mix (Kent Marine Inc., Acworth Georgia 30102, www.kentmarine.com) to every 10 gallons of R/O water to provide soft water hardness reconstitution. A high dissolved oxygen reading can be met by providing adequate filtration and water movement. Kh and Gh (carbonate hardness and general hardness) of water can be tested with simple aquarium grade test kits or with a conductivity meter. (Follow directions for specific brand used).

Monitor ph and ammonia levels as well as nitrate and nitrite levels frequently – daily to weekly depending on stocking levels and age of system set up. Currently we use the Hache water testing system for all water quality tests except nitrate on a daily basis. However, use of simple aquarium test kits for this process (API Freshwater Testing Kit components are sometimes used–

Ammonia, Nitrite, Nitrate, pH, water hardness (Gh and Kh), and Phosphate) work well for retesting water or for quick checks of water quality. Phosphate testing is important, especially in cities with aging pipes or fluctuating water sources. Water should be tested for phosphate

BEFORE it is used in frog tanks. We test for phosphate in water to be used the next day after the reservoir tanks are filled at the end of the day. That way we insure that water is safe for the next day’s use.

The importance of appropriate water for raising young amphibians cannot be overstated. Larval development and metamorphosis are incredibly complex and demanding life stages for amphibians. In addition to diet, some dissolved substances in the water provide nutrients for growth of the larval amphibian. Conversely, some dissolved substances are toxic and create metabolic demands that can interfere with normal growth and metamorphosis.

Changing schedule

All established holding containers should ideally be cleaned 2-3 times per week by siphoning off a minimum of 20 percent and a maximum of 50 percent of the water in the adult frog tanks and replacing it with reconstituted R/O water of similar temperature. Water changes may be daily for larvae holding containers depending on water quality readings.

Chiller units and parts should be checked monthly or as needed and maintained according to the manufacturers guidelines.

Sponge filters should be rinsed out using either fresh R/O or de-chlorinated clean water.

Siphoned out tank water will work as well as this will help maintain the beneficial bacteria growing within them. This should be done as needed or at least weekly.

The frequency of water changes will depend on the stocking density of larvae and the type of filtration system used.

Never use just straight, un-mixed R/O water for frogs or tadpoles.

Charcoal in filters should be changed at least every 6 months or more frequently as needed. (If medications are used then charcoal filters may need to be temporarily removed from system – check with medication instructions prior to use).

For tadpoles, metamorphosed frogs, and adults siphon or skim out all old, uneaten food and feces every day.

Water Issues

Tap water that is aged or allowed to sit for 24 hours is not recommended as most municipal water supply contains Ammonia and Chloramines that are used as disinfectants. If tap water is to be used it must first be treated with dechlorinating and ammonia removing chemicals prior to use in any frog enclosure. Kordon brand NovAqua and Amquel should be used (available at www.drsfosterandsmith.com for example). However, these products can reduce the pH levels so must be monitored for use. Reverse Osmosis cleaned water which is then reconstituted is recommended as carbon filters can be placed in-line to assist with chlorine and chloramines removal. SF Zoo uses only Reverse Osmosis cleaned water (R/O) that is then reconstituted and each room has its own R/O system for water production.

All filters need to be changed regularly, either according to manufacturer’s guidelines or more frequently depending on stocking densities (1x per week to 1x per month)

All nets, siphons and other equipment should be disinfected with a Chlorine Bleach and water mix (1 part bleach to 10 parts water). Equipment will be disinfected away from animal housing areas and will be thoroughly rinsed.

Water should be tested twice weekly with test kits to make sure the filters are functioning properly (chlorine, pH, Kh, Gh, ammonia, nitrite, nitrate, etc) and to look for fluctuations in water quality

During a water change, replacement water should be the same temperature as the water in the holding tanks to minimize stress on tadpoles and frogs. Ideally the water should be held overnight to reach room temperature.

Monitoring Frog Health

Daily counting and health checks must be performed to monitor frog health. Should a tadpole or frog die please collect the animal for pathology at the Zoo Hospital. Note the tank collected, accessioned group or individual number, and animal number (if known for froglets and known for adults).

Frogs should be weighed quarterly to monitor their health and to check operation of pit tags. At each weight period the frogs should be compared with their previous weights to aid in detecting any signs of illness. In addition, all frogs need to be visually inspected during routine husbandry tasks to identify any maladies which the frogs may be suffering from. Any sick frogs should immediately be examined by veterinary staff.

Tadpoles should also be visually examined regularly to detect any signs of abnormal behavior such as swimming upside down or in awkward circles.

Sick and injured frogs and tadpoles in should be immediately transferred into a quarantine enclosure until they have been observed by veterinarian staff.

All YLF postmetamorphic frogs that are wild caught will go through 10 days of itraconazole treatment for chytrid management and prevention of the spread of the disease. Historically all juveniles and adults that have been brought into the Zoo collection have had high levels of Bd that have cleared to zero after treatment. Frogs are tested pre and post treatment and considered positive until a negative status is reported. Equipment, tanks, water and all testing equipment should be clearly marked and kept separated until negative status is reported.

Hibernation and Breeding

Hibernation experiments conducted by the San Diego Zoo indicate that artificial hibernation is an important component of successful breeding in the Mountain Yellow-Legged Frog (Rana muscosa). Hibernation is important in captive frogs to prevent complications from gravid females who may not be able to naturally oviposit eggs without hibernation.

Adult frogs should be hibernated for approximately 90 days at a water and ambient temperature of 4°C (40°F). The frogs should be slowly brought down to this temperature by adjusting temperatures from 13°C down to 4°C one degree per day or every other day. This can either be achieved by cooling the entire room in which the frogs are housed and utilizing chillers to cool the water or by setting the frogs up in refrigerated units with an adequate air flow. Lighting sources shall be eliminated during the hibernation period. Males and females will be hibernated separately to control breeding behavior.

While in hibernation the water quality of the frog’s enclosures should be continued to be monitored at least weekly (ammonia only is necessary) and the frogs health should also be monitored weekly via visual inspections. During water changes it is essential that the temperature of the new water matches the current water temperature and separate water holding systems will need to be in place to provide appropriately chilled water.

At the end of the 90 day period the frogs should be slowly brought up to a temperature of 13°C with a light cycle of three hours per day by increasing the temperature by one degree per day or every other day. Frogs can be fed after being held for one week at 13°C (55°F).

After being brought out of hibernation each frog’s body condition will be assessed by obtaining weight measurements. Male and female frogs should be paired together immediately after hibernation. Eggs should be removed from breeding enclosures 24-48 hours after being laid in order to allow eggs to plump up with water prior to moving to egg holding.

General timeline of breeding and development process:

Males and females brought to room temperature or 55°F and moved into individual pairs.

At 1-2 weeks post-pairing egg masses should be produced.

After eggs are laid, wait 24-48 hours to move so that the eggs can absorb water and expand prior to moving. Transfer the masses using a spatula or soft net.

Egg masses hatch and tadpoles emerge at 14-21 days, generally, when housed at

55-60°F. However, monitor egg masses closely after one week to check for development and separate those that aren’t developing or become moldy.

Tadpole period is usually 3 to 6 months but may last longer depending on temperature, genetics, etc.

Move metamorphing tadpoles out of tadpole tubs when they have 4 limbs. They should be transferred into shallow water with haul out areas.

Tail absorption usually takes 5-7 days. When the black nub is all that is left of the tail start to offer food items to metamorphs though they generally won’t eat for up

7 days post morphing. Food items should be ½ the width of the mouth when viewed on the B-axis.

Appendix I – Water Testing

Directions for Water Testing with API Test Kits pH

1. Rinse a clean test tube with water to be tested

2. Fill test tube with 5 ml of aquarium water (to the line on the test tube)

3. Fill test tube with 5 ml of water

4. Add 3 drops of pH solution

5. Cap tube and invert tube several times to mix

6. Determine pH by matching color to pH chart

7. Rinse tube with clean water

8. pH levels should be 6.5 - 7.2

Image from Aquarium Coop

Ammonia

2. Fill test tube with 5 ml of aquarium water

3. Add 8 drops of Ammonia test solution #1

4. Add 8 drops of Ammonia test solution #2

5. Cap tube and shake tube vigorously for 5 seconds to mix

6. Wait 5 minutes for color to develop

7. Determine Ammonia levels (ppm) by matching color to chart

8. Rinse tube with clean water

9. Ammonia levels should be < 0.25ppm (< 0.02 mg/L) (over 0.5ppm could be deadly)

Nitrite

2. Fill test tube with 5 ml of aquarium water

3. Add 5 drops of Nitrite solution

4. Cap tube and shake tube vigorously for 5 seconds to mix

5. Wait 5 minutes for color to develop

6. Determine Nitrite levels (ppm) by matching color to chart

7. Rinse tube with clean water

8. Nitrite levels should be <0.5ppm (< 50 mg/L) (over 1.0ppm could be deadly)

Nitrate

2. Fill test tube with 5 ml of aquarium water

3. Add 10 drops of Nitrate test solution #1

4. Cap tube and invert tube several times to mix

5. Vigorously shake Nitrate test solution #2 for at least 30 seconds

6. Add 10 drops of Nitrate test solution #2

7. Cap tube and shake tube vigorously for 1 minute to mix

8. Wait 5 minutes for color to develop

9. Determine Nitrate levels (ppm) by matching color to chart.

10. Rinse tube with clean water

11. Nitrate levels should be < 20ppm (< 1 mg/L)

Gh – water hardness

2. Fill test tube with 5 ml of aquarium water

3. Add General Hardness Test Solution, ONE DROP AT A TIME counting the number of drops.

4. After first drop is added, solution will turn orange. If the water sample contains only

1°dGH, the solution will turn from clear to its green endpoint after the first drop is added.

5. Cap the test tube and invert several times after each drop. Keep count of the drops being added.

6. The test is completed when the water in the test tube, after having been shaken, turns from orange to green. If you have difficulty discerning the color after the first drop of test solution is added, remove the cap from the test tube and while holding it over a white background, look down through the tube.

7. The General Hardness value is determined by the number of drops of the reagent that must be added to turn the water in the test tube green. Each drop is equal to 1°dGH or

17.9 ppm GH (see the chart).

8. Rinse tube with clean water.

9. Water hardness should be very soft to match water from Sierras with Gh values of 50-75 mg/L.

Kh – water hardness

2. Fill test tube with 5 ml of aquarium water (to the line on the test tube).

3. Add Kh Carbonate Hardness Test Solution, ONE DROP AT A TIME.

4. After first drop is added, solution will turn blue. If the water sample contains only

1°dKH, the solution will turn from clear to its yellow endpoint after the first drop is added.

5. Cap the test tube and invert several times after each drop. Keep count of the drops being added.

6. The test is completed when the water in the test tube, after having been shaken, turns from blue to yellow. If you have difficulty discerning the color after the first drop of test solution is added, remove the cap from the test tube and while holding it over a white background, look down through the tube.

7. The Carbonate Hardness value is determined by the number of drops of the reagent that must be added to turn the water in the test tube bright yellow. Each drop is equal to

1°dKH or 17.9 ppm KH, (see the chart).

8. Rinse tube with clean water.

9. Water hardness should be very soft to match water from Sierras

Phosphate (PO4)

2. Fill test tube with 5 ml of aquarium water

3. Add 6 drops of Phosphate test solution #1

4. Cap and invert tube

5. Shake vigorously for 5 seconds to mix

6. Add 6 drops of Phosphate test solution #2 (NOTE: bottle #2 contains very thick solution and may require increased pressure to release drops)

7. Cap tube and shake tube vigorously for 5 seconds to mix

8. Wait 3 minutes for color to develop

9. Determine Phosphate levels (ppm) by matching color to chart

10. Rinse tube with clean water

11. Phosphate levels should be 0 ppm

Chlorine

1. Rinse a clean test tube with water to be tested.

2. Fill test tube with 5 ml of aquarium water

3. Swish a Cl test strip in the tube for 10 seconds.

4. Remove test strip and wait 10-20 seconds.

5. If there is a color change on the test strip, compare to color chart on the bottle.

6. Rinse tube with clean water.

7. Chlorine levels should be undetectable (no color change).

Temperature

1. Look at room temperature. Temp should be 50-65°F.

2. Look at and record water temperature on health sheet.

Image from Aquascaping Love wedsite

Appendix II - Pathology Submission Protocol for Dead Yellow-Legged Frog

Tadpoles and Postmetamorphic Frogs

All YLF tadpoles and postmetamorphic frogs that die will be submitted to the Zoo veterinary hospital for complete necropsy examination and final carcass disposition. Complete necropsies that include histopathology of all major organ systems are essential for detection and control of important infectious disease agents or other disease problems that could be related to captive husbandry or nutrition.