1 hour

Condom Negotiation: Strategies for Responding to Pressure 1 hour Saving and Managing Money: Snakes and Ladders Game
By the end of this session, participants will be able to: Identify a goal and ways to save money to reach that goal
More on HIV: Immune System, Disease Progression, and Living Healthy
By the end of this session, participants will be able to: Understand the effect of HIV on the body and how it progresses Explain the difference between HIV and AIDS Explain three ways to prevent mother-to-child-transmission of HIV List positive behaviors that can keep a person who is HIV+ or who has AIDS live healthy longer By the end of this session, participants will be able to: Identify the relationship between alcohol and risk behavior
Alcohol and Sex Work: Spin the Bottle
Curriculum for Commercial Sex Workers (Continued)
Session Name Summary of Objectives By the end of this session, participants will be able to: Understand what it means to feel at risk for HIV and make decisions about getting tested Understand a typical C&T session Identify options for getting tested locally
Counseling and Testing (C&T): The Decision to Test

Living with HIV

By the end of this session, participants will be able to: Describe what it means to live with HIV Understand the effects of stigma and discrimination on people living with HIV Describe how their attitudes towards people living with HIV have changed after the session
Confronting Stigma and Discrimination
By the end of this session, participants will be able to: Discuss different types of HIV-related stigma and discrimination Understand some of the underlying causes of stigma and discrimination Suggest strategies for challenging stigma in their own contexts

HIV/STI Risk

By the end of this session, participants will be able to: Clarify how HIV/STIs can be transmitted and can not be transmitted. Consider ones own risks of HIV/STI infection. By the end of this session, participants will be able to: Practice responding to situations that may make them vulnerable to HIV infection Share strategies for responding to these situations By the end of this session, participants will be able to: Visualize their preferred futures Create a plan for the future
Making Yourself Less Vulnerable to HIV (Situation Role Plays) 2 hours Planning for the Future & Closing 2 hours
You may decide to organize groups differently based on your knowledge of the target groups and local culture.
2 the Road to Good Health: HIV Prevention in Infrastructure Projects
Training Schedule for Sex Workers
Length of time (approximate) 1 to 2 hours Welcome & Opening HIV Transmission and Prevention More on HIV: Disease Progression and Living Healthy Confronting Stigma and Discrimination HIV/STI Risk Cards Monday Tuesday Wednesday

Activity II. Introductory Skit (Approximately 15 minutes)
1. INDICATE that we will begin with a discussion of how HIV is transmitted, the basics of how to prevent it, and a look at when we might be most at risk for HIV infection. 2. Without any further explanation, DIRECT participants attention to the front of the room, and step to the side as the group silently performs the introductory skit. 3. After the performers move off of the stage, PROCESS the skit using some of the following questions: What happened in the skit? What struck you? What stands out from the skit? What did the clinking represent? The
6 the Road to Good Health: HIV Prevention in Infrastructure Projects
exchange of water or tea? What was going on with her (refer to the sex worker who always used condomsFemale Sex Worker #1)? What was she doing? Why did she not get infected? What was the basic behavior of (point to Female Sex Worker #1)? How did she get HIV? Describe the behavior of the (refer to the HIV+ woman). Did she pass HIV on? and so on. 4. SUMMARIZE the discussion, briefly describing some of the situations of vulnerability raised by the skit. Invite participants to talk a bit about the situations that make sex workers vulnerable to HIV transmission. Trainer Tip This exercise has been used successfully with groups from many cultures all over the world, including East Asia. Participants enjoy performing it and there are many advantages to engaging workshop participants in the skit. An option is for facilitators and others involved in the program to serve as the actors with the one advantage that they can prepare and practice earlier. Key Points: Emphasize that it is not our role in life that dictates whether or not we are vulnerable to HIV. For example, Female Sex Worker #1 was careful to use condoms every time with her clients, but thought that it was safe not to use them with her boyfriend. Her boyfriend was having sex outside of the relationship without a condom, so they both got infected anyway. Be sure to discuss the issue of men offering more money to sex workers for sex without a condom. Discuss the inherent dangers in this practice, using the role play as an example. Point out that the woman who began the skit as HIV-positive chose not to engage in relations with anyone and did not transmit HIV to anyone. Use this as an opportunity to talk about stigma against those of us living with HIV. Some people fear those of us living with HIV because they think they can get HIV from us, but often, those of us living with HIV have become better informed and protect ourselves well. (Emphasize that one has to know that one is infected for this to be true.) Emphasize that the only actors in the skit who did not get infected with HIV in the skit were those who chose either to abstain from sex, or those who chose to protect themselves when having sex. Remind the group that we will talk more about protection in a bit. 5. Be sure to DE-ROLE the actors, and to emphasize that the skit was only intended as an example of risk and vulnerability. It was not intended to say that all sex workers are HIV positive, that all men who party are HIV positive, that all construction workers have sex with sex workers, and so on.

Worksheet: Condom CarouselSample Questions
How many times can you use a condom? Male condoms can only be used once, and they should never be reused. It is recommended that female condoms also be used once. But in resource-poor settings, it is possible to wash the female condom and use it again. A protocol for properly cleaning the female condom is available from the Centers for Disease Control. Petroleum jelly (i.e. Vaseline, or local brand) is a good lubricant to use with a condom. True or false? It is not safe to use any oil-based lubricant with a condom (i.e. petroleum jelly, Vaseline, and so on). Water-based lubricants should be used, such as K-Y Jelly or (insert local brand). It is not safe to use two condoms at the same time. The friction between the two condoms makes it more likely that the condoms will break. Also, using two condoms may be so uncomfortable, it may reduce the likelihood that you will use condoms again. One condom, properly used, is the best protection against HIV infection. False. Condoms do not transmit HIV. They offer good protection against HIV transmission. (In some countries, there have been rumors that HIV is actually inside condoms.) Couples can use condoms to prevent HIV and also to prevent unwanted pregnancy. Unless the couple is trying to conceive a child, condoms are a good choice of family planning that offer the added protection against HIV and other STIs.
Is it safer to wear two condoms instead of just one, or a male and female condom at the same time? Is it double the protection?
Condoms sometimes transmit HIV. True or false?
Why is it good for married couples to use condoms, or to use a condom with your boyfriend?
Session C: Condoms with Trusted Partners
Objectives By the end of this session, participants will be able to: Realize that a partners past behavior may put you at risk even if his current behavior is faithful Understand that condom use is important even with trusted partners Trainer Tip Session is adapted with the permission of Family Health International: Learning about Healthy Living: An Activity Manual for Outreach Workers, Family Health International/ USAID, 2007. Can be accessed at: http://www.fhi. org/en/HIVAIDS/ pub/guide/res_ HealthyLiving.htm. Time Materials Approximately 1 hour Small photos of 10 beautiful women and 10 handsome men (film stars work well) Small papers cut the same size as the photos (10 blue and 10 green) Preparation Buy the small photos or make them by cutting pictures from magazines. Place an X mark on the back of three of the pictures. (NOTE: Do not let participants see the back of the cards. At the end of the activity, the cards will be turned over and the X will indicate which person had HIV and will indicate how HIV may have been transmitted to the other partners.)
Activity I. Telling Our Stories
1. WELCOME the group. SHOW the female pictures. 2. ASK a volunteer participant to choose the picture that she wants to represent herself. 3. ASK the participant to choose picture(s) of men who can represent her current boyfriend(s). 4. TELL her to place the male picture(s) next to the picture that represents herself. 5. ASK: Have these men had other partners in the past or do they have sex with other people now? The participant should select pictures of the women that her partner has had or does have sex with. If she has actually seen the other women, she can select a photo to represent that woman. If she knows about the other sex partner (e.g., the wife) but has never seen her, she should just add a green card and place it next to the man. 6. ASK: Have these female partners had other partners in the past? If so, place either photos or blank blue cards next to the females. 7. CONTINUE asking about previous partners of the main partner or of the part-

Introduction (Approximately 5 minutes)
ASK: Why do you want to save money? What is your goal for saving money?
EXPLAIN: In this game, you will reach your own personal goal if you can reach square # 100.
Activity I. The Game (Approximately 55 minutes)
PLAY: 1. participants choose their markers (each a different color or shape.) 2. All participants start at square 1. 3. Participants take turns rolling the dice (or the pencil) and moving markers the number of squares that appears on the dice. 4. If a marker lands on a square that has written instructions, the participant must follow the instructions and either go forward toward her goal or fall backwards away from her goal. 5. The first participant to reach square # 100 wins. 6. SUMMARIZE THE GAME: 7. ASK: Are there some things in this game that are like your life? If you saved money would you be able to go home more often? Can you think of things to do for entertainment other than going to a beer shop and visiting with women? How might this increase your chance of protecting your family [from HIV].

Game Board

Session F: Alcohol and Risk: Spin the Beer Bottle
Objectives By the end of this session, participants will be able to: Identify the relationship between alcohol and risk behavior Identify ways to provide peer support to not drink in excess. Time Trainer Tip Session is adapted with the permission of Family Health International: Learning about Healthy Living: An Activity Manual for Outreach Workers, Family Health International/ USAID, 2007. Can be accessed at: http://www.fhi. org/en/HIVAIDS/ pub/guide/res_ HealthyLiving.htm. Materials Approximately 1 hour Cards (about 15) to write short statements on An empty beer bottle Preparation Decide on appropriate statements for your audience and write one on each card. Decorate the back of the cards with fun pictures. Suggestions include the following: Drinking a lot of alcohol can harm your health. True or False? Act out typical behavior of a drunk customer. Why do people like to drink so much alcohol? Are there benefits of having sex while sober? What are they? Tell at least two ways to prevent HIV transmission. What is the difference between having HIV infection and having AIDS? How can you trick a drunk client into using a condom? How can you sell a lot of beer to clients without getting drunk yourself? Put a condom on the beer bottle using your mouth. Will you go on a motorbike with a drunk customer? Why or why not? Spin the bottle again.

Activity II. Elephant and Lions Game (Approximately 15 minutes)
1. SUGGEST that we will do a short activity to get us started. This activity can give us a simple way to remember the impact of HIV on the immune system. 2. INVITE one volunteer to join you in the center of the semi-circle. Tell the group that this person is the baby elephant. 3. ASK for six more volunteers. These volunteers are the adult elephants. Their job is to protect the baby elephant. They should form a circle around the baby elephant, facing out, away from the baby elephant, and link arms or join hands. 4. Now, ASK for four or five more volunteers. These people are the lions. Their job will be to attack the baby elephant. 5. INDICATE that when you say GO! the lions should try to attack the baby elephant. Let this go on for about 10 secondsuntil the baby elephant has been shoved or touchedbut dont let her get hurt! 6. PROCESS this part of the exercise with the group, perhaps by asking some of the following questions:
Possible Questions: What is the baby elephant? What does it represent? What are the adult elephants? What do they represent?
Possible Responses: The baby elephant represents the human body. The adult elephants represent the immune system. Their job is to protect the baby elephant, just as it is the job of our immune systems to protect our bodies from disease.
Possible Questions: What are the lions? What do they represent?
Possible Responses: The lions represent the diseases or infections that try to attack our bodies often. We are often exposed to germs and organisms that invade our bodies and make us sick. The immune system tries to fight off these germs and organismsto keep us healthy, or to help us to get healthy once we are sick. Usually not, as the immune system protects the body and helps us to get better after an illness.
Even though the lions or diseases attack the human body, are they able to kill it?
7. Next, MOVE dramatically from one of the adult elephants to another, and say, But suppose I am HIV. I come to this body and begin to attack and kill the immune system. Remove a few of the adult elephants by asking them to sit down. Leave only two adult elephants, then ask the group, What will happen to the baby elephant when the lions attack now?
Key Points HIV is yet another germ or invading organism. It comes into the body and attempts to infect it, or make it sick. But HIV is different from other organisms in important ways. Usually the immune system protects the body and fights the invader, but HIV attacks the immune system, making it weaker. So, HIV makes it easier for other diseases and illnesses, like diarrhea, tuberculosis, and so on, to attack and hurt the body. 8. Once more, GIVE the signal for the lions to attack, but only for a few seconds, to show the impact of the diseases on a weakened immune system. 9. PROCESS the exercise and be sure to review the key facts. Then, thank all participant volunteers and segue immediately into the next session.

Trainer Tip Remind the group that a basic understanding of this is helpful, especially for those trying to understand how to live healthily with HIV, and to help us to understand the best ways to fight the virus. It is not necessary to remember all of the facts about the immune system and so forth, and we are omitting a great deal of information about how the virus works, and technical terms for the immune system. Indicate that you are available after the sessions to give resources for further information, for anyone interested in more detail.

Antibodies

Viral Load X Window Period Honeymoon Period T i me AIDS

Antibodies Symptoms

8. Finally, GUIDE the group through a discussion of the transition from HIV to AIDS. Indicate that after a time, the antibody response may begin to fail, and ones viral load may begin to climb again. There are only so many immune system cells that one body can generate in a lifetime, and the immune system begins to fail after a time and loses its battle against HIV. At this time, the viral load climbs, and the antibody response drops. (Draw a declining line with the blue marker and a rising line with the red marker.) Ask participants if they know what this time in the life of someone living with HIV is called. Invite the participant with the AIDS card to add it to the diagram. 9. Indicate that at this time, when other diseases or illnesses attack the body, the immune system is unable, or less able, to respond. A disease that otherwise would not do much damage, like diarrhea, may now be capable of making
that person seriously sick. Remind the group about the same situation in the Elephants and Lions exercise. When someone has AIDS, these diseases can now attack and even kill the person, just like the lions were able to attack the baby elephant once the mother elephants were eliminated. 10. CHECK for participants understanding of HIV disease progression, by asking a few questions: Questions What is the difference between HIV and AIDS? Can someone explain it? Possible Answers HIV is the virus that enters the body and attacks the immune system. When the antibody response begins to fail and the amount of HIV in the body climbs, the person develops Acquired Immune Deficiency Syndrome, or AIDS, which is a syndrome of diseases that continue to make the person sick and may eventually lead to the persons death. No, people do not die of HIV. As a matter of fact, people can live a relatively long life with HIV with proper treatment and care. People die of AIDS, which develops if HIV is able to defeat the immune systems antibody response against HIV. The drugs that we think of as HIV drugs or AIDS drugs are called ART, or antiretroviral therapy. These drugs fit in during the late part of the honeymoon period, or just at the beginning of the time when someone develops AIDS. These drugs help the immune system to fight HIV, and they can boost the amount of antibody response, and drop the HIV viral load to minimal or even undetectable levels. They can, in effect, keep people in the honeymoon period for years or decadeswe dont yet even know how long they can keep someone alive and healthy living with HIV.

Can someone die of HIV?

Trainer Tip Emphasize that in many locations, cesarean birth is not safe enough for this to be an alternative, and a natural birth is recommended. Similarly, in many areas, it is not safe to use alternatives to breast milk, due to cost, availability, or contaminated water. In these cases, breastfeeding is still recommended.
What about these new HIV drugs? Where do they fit on this diagram?
Questions How might a mother prevent motherto-child transmission of HIV?
Possible Answers First, she must know that she is infected with HIV, so she should be tested at the beginning of her pregnancy (if not before) to find out her HIV status. Next, there are specific antiretroviral drugs that she can use before delivery, and that can be given to her infant after delivery, to help keep the baby HIV-negative. Also, it is best if she is able to give birth by cesarean birth, if that is safe and available, as it limits the amount of blood and amniotic fluid the baby is exposed to during labor and delivery, which is the time when most infants become infected. Finally, if alternatives are safe and available, she should not breastfeed the infant, as HIV can be passed through breast milk.
11. SEGUE immediately to the Living Healthy segment, by reminding participants that the honeymoon period is perhaps the most significant part of disease progression. The honeymoon period can be a time of hope for those infected with HIV, as it offers the opportunity to remain healthy for a longer period of time, and to delay the onset of AIDS.
Activity IV. Living HealthyThe Well-being Wheel (Up to 30 minutes)
1. POST the Well-being Wheel flipchart directly over the honeymoon period in the diagram. 2. REVIEW the five aspects of well-being on the chart. Take each in turn, and brainstorm possible interventions for each of the five segments on the wheel. General Well-being Good nutrition and sanitation; Rest and exercise; Avoiding smoking, drugs, alcohol; Avoid STIs and reinfection with HIV Immune system enhancers; Traditional herbs; Acupuncture; Prompt treatment of opportunistic infections; Treatment with antiretroviral therapy

Physical Well-being

Social Well-being
Support of ones spouse or partner; Extended family support; Peer support; Productive work; Advocacy work; Protection from discrimination Counseling; Positive attitudes; Stress reduction; Interpersonal skills-building; Self-esteem building Faith; Meditation; Belief system; Prayer

Psychological Well-being

Spiritual Well-being
3. SUMMARIZE and CONCLUDE the session.

Good nutrition

Immune System Enhanced
General Physical Well-being Well-being Faith Meditation Prayer Belief System Psychological Well-being Counseling Stress reduction Self-esteem building Spiritual Well-being Social Well-being Support of Spouse/partner Family Support Peer Support

Write down one secret or confidential piece of information about yourself that no one or few people know about. It might be something very private to you, which you would prefer no
one finds out. Write down this secret or personal piece of information on #4. Indicate that participants may write it in code if they are very worried someone will see their answers. Lastly, write down the name of the person whose love and support means the most to you in the world. 6. After participants have finished, EXPLAIN that an accident or tragedy has just happened in their lives. Because of this situation, they will be forced to lose two of the items in their hands. They can choose which two things they will lose. They might also choose to have their secrets exposed. Indicate that you will come around the room and collect the two cards that they choose. 7. MOVE around the room with the bowl, and collect two cards from each participant. 8. ALLOW a few silent moments for the participants to experience the emotions associated with these losses. Some participants may be a bit upset or uncomfortable at this point. 9. ASK participants to describe in one word or phrase the emotions they are feeling. Write the words on a blank flipchart. Keep brainstorming until all of the ideas are exhausted. 10. Next, INDICATE that this same tragic occurrence will lead to additional losses, and participants may not always have control over these changes in their lives. Indicate that you will now go around the room and randomly take cards from participants. 11. MOVE around the room with the bowl, and randomly take cards from participants. You might take all cards from some, and take no cards from others, while you might take 1-2 cards from others. 12. ALLOW a few moments for participants to process these new losses. 13. ASK participants to describe in one word or phrase the emotions they are now feeling. Write the words on another section of the flipchart. 14. INVITE participants to look at the lists they have created. Ask them to imagine how these feelings might relate to testing positive for HIV. Discuss the potential links between this exercise and testing positive, with special emphasis on the feelings of loss, fear, stigma, isolation, and so on. 15. Be sure to ELABORATE on the fact that as in the first part of the exercise, people living with HIV may have control over some losses to suffer as a result of their status (for example, they may decide to lose a certain amount of comfort by refusing to disclose their status to close family and friends, or they may decide to give up some particular activity in order to afford treatment, etc.). Other losses, however, might be randomly taken away (for example, their rights to property or to work might be taken due to HIV-related discrimination). 16. GUIDE a discussion around what this might mean for the support that they

Activity II. Defining Stigma (20-30 minutes)
1. REMIND the group of the discussion around C&T. Suggest that perhaps the most powerful deterrent to C&T and to disclosing status is the stigma and discrimination that people fear they will be subject to in their homes and communities. 2. SUGGEST that it is often said that there are three
Trainer Tip As commercial sex workers, participants may already be exposed to a great deal of stigma and discrimination. You may wish to guide a discussion about how the stigma that they are already coping with is similar to the stigma suffered by those of us living with HIV. What strategies have we used to cope with stigma against commercial sex workers? How might this stigma and discrimination be compounded by testing positive for HIV?
phases to the AIDS epidemic in any society. The first of these is the epidemic of HIV infection. This enters a community silently and unnoticed. Next follows the epidemic of AIDS, which appears when HIV triggers life-threatening infections. Finally, there is the third epidemicthe epidemic of stigma, discrimination, blame and collective denialthat makes it so difficult to effectively tackle the first two. 3. ASK participants to think about instances of stigma and discrimination around the topic of HIV in their own lives, in their communities, in their countries. Ask, What have you seen that strikes you as stigma? 4. PROCESS the stories, by asking the following questions. (You may wish to ask a co-facilitator to write the answers on a flipchart.) What are some of the forms of stigma that you saw in the stories? What does it look like? (Name calling, scapegoating, ridicule, shaming, blaming, judging, gossiping, not sharing food or utensils, blaming and isolating oneself, stigma by association, and so on) What might be some of the causes of stigma? Why do people stigmatize others? (Morality; peoples beliefs about contagion or impurity; fear of infection; fear of the unknown; fear of death; ignorancelack of knowledge and misconceptions; gender and poverty; prejudice, and so on) What might be some effects or consequences of this stigma? What is the impact of stigma? (Shame, denial, self-isolation, loneliness, neglect, loss of hope, depression, self-blame, selfpity, anger, loss of opportunities, and so on)
Trainer Tip This part of the session is optional. You may wish to remove it, or to simply describe the difference between stigma and discrimination. It is very important to emphasize here that two of the most hurtful consequences of stigma are 1) those who need access to services like C&T, treatment, and so on may not feel safe to seek access to these servicesthis may result in becoming sick or even dying as a consequence of stigma, and 2) prevention of HIV is hampered, because HIV becomes too stigmatized a topic to address.

Dont stand too close to someone with HIV
I feel sorry for the children who get HIV, because they are innocent victims. If I got HIV, Id kill myself.
I dont want my children to go to school with a child who is HIV positive. She looks so thin, she must have AIDS.
Sample statements In the workshop, several AIDS victims came to speak to us
Key Points This language is stigmatizing because calling those of us living with HIV victims makes us sound powerless and weak, when in fact, we can be strong and healthy even with HIV infection.
**The Hot Seat Exercise was taken from: Understanding and Challenging HIV Stigma: Toolkit for Action, Change Project, Academy for Educational Development and International Center for Research on Women, 2003, p.139. Can be accessed at: http://www.icrw.org/docs/2003-StigmaToolkit.pdf.
Session L: Making Yourself Less Vulnerable to HIV
Objectives By the end of this session, participants will be able to: Practice responding to situations that may make them vulnerable to HIV infection Share strategies for responding to these situations Time Materials Approximately 2 hours Prepared situation cards
Preparation Prepare situation cards prior to the session. It is important to choose situations that are relevant to participants and that resonate for them.
Activity I. Introduction and Set-Up (Approximately 15 minutes)
1. WELCOME the group. Indicate that we have shared a great deal of information about HIV. But we know that information is not enough to keep people from contracting HIV or other sexually-transmitted infections. Vulnerability to HIV may have more to do with knowing how best to respond to a difficult situation, or having the resources to get out of a difficult situation. Trainer Tip If the group is large, you may wish to divide into smaller groups but you will need to make sure that you have one facilitator to work with each group. 2. TALK a bit about the situations that make participants vulnerable to HIV infection. Indicate that it can be helpful to think about and create strategies to address these issues before participants find themselves in these situations. This session is intended to provide an opportunity to create and share such strategies, and to practice responding to certain situations. 3. PROVIDE instructions for the activity. We will sit in a circle. In turn, the facilitator will choose 2-3 people from the group for a practice exercise. These people will be given small situation cards. They will quickly read the card and act it out. Try to act as realistic to the situation as possible, and try to implement strategies to make you less vulnerable in the

situation. Each situation will take about 5 minutes, and then new participants will be chosen for another situation. Check for participants understanding of the instructions. Before beginning, remind the group that this is their opportunity to practice and integrate the many bits of different information they have shared during our time together. Urge them to really use this time to learn, practice, and apply their new learnings.
Activity II. Practice in Reducing Vulnerability (Remainder of the Session)
1. BEGIN the exercise by choosing participants for the first situation, and handing them their cards. You may need to guide the first one a bit to ensure participant understanding of the process: Choose participants to serve in the situation. They should come into the center of the circle, either standing or sitting in chairs that they bring into the circle. After the situation has concluded, provide time for participants in the outer circle to offer feedback and suggestions or to ask questions and clarify information. Offer advice and guidance about possible approaches to the situation. Begin the next situation, by choosing new participants for the center of the circle. Continue in this way until all situations have finished. Be sure to flesh out any resources available. 2. When all role plays have finished, RECONVENE the large group. PROCESS the activity: Invite participants to offer feedback about the activity. What are their impressions? What stands out for them? What particular situation(s) caught their attention the most?
If time, briefly review each of the role play situations and invite feedback from participants regarding how they recommend dealing with the situation. Be sure to emphasize the human rights of each person, regardless of sex or profession, to determine what happens to their body. Discuss the rights of sex workers to decide how and when they will have sex.
3. SUMMARIZE the discussion.
You are a sex worker, and you usually use condoms with your clients, except for one or two of your regulars. You are with your boyfriend right now, about to have sex, and you suggest that he use a condom.

Activity I. My Life Story (Approximately 45 minutes)
1. WELCOME the group back, and remind them that this will be our last session together. Summarize our time together. 2. REMIND the group that we have been talking HIV prevention, and protecting ourselves so that we may reach the goals we have set out for ourselves. Weve strategized about how to save money to better reach our goals. In this final session, we want to take some time to visualize our desired futures, so we can keep this vision in mind and let it take us closer to the realization of these goals.
3. INVITE participants to sit back and close their eyes. Invite them to imagine what the future holds for them. At the end of their lives, what story do they most want to tell? What is their preferred future? 4. INVITE participants to meet in pairs and share their life stories. Encourage
them to start at the beginning, and to talk about how they moved from where they are now to the realization of their goals and dreams. 5. ALLOW about 15 minutes for the first life story, then remind the partners to switch, so that the other partner can tell her life story, too.
Activity II. The Road Mapa Planning Exercise
1. RECONVENE the group. INDICATE that we are now going to do a bit of planning to help us to bring these dreams closer to reality. 2. SKETCH a sample road map on the flipchart. (Be sure to make a joke about building roads together) Begin by drawing a box, circle, house, etc. at the far right, and put in the various components of the happy ending or the dreams that the people in the group might have. (Examples might include having a healthy family, having a house or business of their own, and so on.) 3. Next, SKETCH a box or circle at the far left, and fill in the current situation there. (For example, working on the roads project, but sending money home to build a house, and so forth.) 4. SHOW participants how to make a road map between the current situation and the preferred future. Along the road, participants should fill in the various steps needed Move to to move from their current bigger city realities to their preferred futures. Also take time to Save money put in bumps or potholes in the roadthings that can Back off drugs take the participants off track from their preferred futures. (Examples may be getting Now - sending addicted to drugs, unwanted money home - Working as CSW STIs, and so forth.) How will they build a bridge to get over those potholes, or make a detour to get around those times?

dependent, whereas male differentiation appears to be dependent on the absence of estrogen (9). Many potential endocrine disrupting compounds are present in snapping turtle eggs and may influence sex differentiation (10). There is evidence that reptiles are sensitive to endocrine disruptors. Slider eggs (Trachemys scripta) exposed to estrogenic PCBs produced females at male-producing temperatures (11). Furthermore, the presence of these organochlorines may disrupt normal endocrine function and morphology in sexually mature reptiles. Female alligators (Alligator mississippiensis), at a site contaminated from a spill of Dicofol and DDT, had abnormally high concentrations of 170estradiol in blood plasma, whereas males had depressed testosterone levels and increased levels of 170-estradiol. Also, juvenile alligators born after the spill had poorly organized testes and small phalli (12,13). Environmental contaminants have also been implicated in demasculinization of Florida panthers (Felis concolor cory:) (14), in lower sex steroid and reduced male secondary sex characteristics in white suckers (Catostomus commersoni) (15), and in feminization of California gulls (Larus californicus) (16). Feminization in turtles may be expressed by changes in the circulating levels of hormones and changes in sexually dimorphic morphology. The precloacal length is longer in male turtles than in females (17 because
this is where the penis is located. We examined the penis of a dissected adult male snapping turtle (carapace length = 33.9 cm); the tip of the penis in the relaxed position was within 1.5 cm from the cloacal opening. The precloacal length of male snapping turtles is usually longer than the two posterior plastron lobes, whereas it is shorter in females (17). In males, the precloacal area grows in length faster than the carapace, whereas in females, these two areas grow at the same rate (17). If exposure to exogenous endocrine-disrupting compounds during sexual development results in feminization, it may reduce the rate of growth of the precloacal length. Consequently, the ratio of the precloacal length to the posterior lobe (PPR) would be smaller in contaminated turtles. We tested the hypothesis that snapping turtles from contaminated sites would be feminized when compared to snapping turtles from noncontaminated sites. We predicted that snapping turtles at contaminated sites would have depressed testosterone levels and elevated estrogen levels in their blood plasma and would have smaller PPRs. An alternative hypothesis is that variation in sexually dimorphic morphology and levels of sex steroids is dependent on abiotic factors such as temperature, length of active season, or other dimactic factors. We tested this hypothesis by determining if latitude could explain the observed differences in testosterone and estrogen levels and sexually dimorphic morphology.

Materials and Methods

Snapping turtles were caught at five different sites in Southern Ontario. Lake Sasajewun (4535'N, 7830'W), which is located at the wildlife research station
Address correspondence to C.A. Bishop, Canadian Wildlife Service, Canada Centre for Inland Waters, 867 Lakeshore Road, PO Box 5050, Burlington, Ontario, Canada, L7R 4A6. We thank Len Simser (Royal Botanical Gardens), David Lean (National Water Research Institute), the Ministry of Natural Resources, and the Central Lake Ontario Conservation Authority for permission to capture turtles, and we thank Holly Spiro, Melinda Portelli, Nicola Koper, and Meg Krawchuk for field work. Funding was provided by the Great Lakes Action Plan, Environment Canada, the Tri-council Eco-Research Program, and NSERC grant A5990 (RJB). Received 6 May 1997; accepted 14 January 1998.
Environmental Health Perspectives * Volume 106, Number 5, May 1998
Articles * de Solla et al.
(WRS) in Algonquin Provincial Park, is a 45-ha dystrophic lake with no history of industrial or agricultural discharge. Snapping turtle eggs from Lake Sasajewun contain very low to nondetectable organochlorine concentrations (5). Jack Lake (4442'N, 7802'W) is a dystrophic lake with numerous cottages along the shores but no industry, and it has only background levels of organochlorine contaminants in plankton (1). These levels are consistent with Ontario lakes that are contaminated primarily through atmospheric deposition (18). Cootes Paradise (43017'N, 7953'W), in Hamilton Harbour, is a 45ha eutrophic wetland adjacent to heavy industry and sewage treatment plants on the shore of Lake Ontario. Lynde Creek Marsh (4345'N, 7857'W) in Whitby, Ontario, is a eutrophic marsh and creek that drain into Lake Ontario. Big Creek Marsh (42 31'N, 80 29'W) is a 615-ha shoreline marsh that drains a large, agriculturally based watershed into Lake Erie. Snapping turtle eggs collected in 1989 from Lake Sasajewun had significantly lower concentrations of total PCB congeners and pp'-DDE [0.32 and 0.04 pg/g wet weight (ww), respectively] than Cootes Paradise (30.57 and 4.52 pg/g ww) or Lynde Creek Marsh (37.57 and 3.62 pg/g ww), whereas eggs from Big Creek turtles had intermediate concentrations (6.23 and 0.74 pg/g ww) (4,5). For this study, Lake Sasajewun and Jack Lake were considered control sites, Lynde Creek Marsh and Cootes Paradise
were designated as contaminated sites, and Big Creek was considered to be a moderately contaminated site.
The morphology of snapping turtles was compared from data collected from Lake Sasajewun in 1987-1991, Jack Lake and Lynde Creek Marsh in 1995, Cootes Paradise in 1994-1995, and Big Creek Marsh in 1986-1987. Snapping turtles were caught with baited or unbaited hoop traps from early May to late August. The traps were left overnight and checked every morning. We trapped 15 female and 5 male snapping turtles at Lake Sasajewun from May 29 to July 22, 5 females and 4 males at Lynde Creek from June 25 to July 7, 14 males and 6 females at Jack Lake from July 25 to Aug 14, and 90 females and 80 males at Cootes Paradise from May 8 to Aug 22. Two observers measured turtles: one at Cootes Paradise, Jack Lake, and Lynde Creek and another at Big Creek and Lake Sasajewun. Because we had separate observers taking measurements at different sites, there was a risk that observer bias could affect our results. However, one of the observers from Lake Sasajewun had measured snapping turtles from Cootes Paradise in 1992, which allowed us to test for this potential bias. We compared measurements from Cootes Paradise between 1992 and 1994-1995. Precloacal length was measured from the posterior of the plastron to the doaca with the tail stretched into a natural position (19). The posterior lobe was measured as the length of the two posterior plastron scutes (Fig. 1). Whenever possible, the males were positively sexed by eliciting eversion of the

penis and females were identified by capturing nesting turtles. In 1995, blood samples were-obtained from adult snapping turtles (carapace length >20 cm) (18) from Lake Sasajewun, Jack Lake, Cootes Paradise, and Lynde Creek Marsh. Blood samples were taken within an hour from when the turtles were removed from the trap, usually between 0900 and hr. Approximately 4 ml blood was taken from the caudal vein using 5-ml lithium heparin vacutainers and 22 gauge double-sided needles. The blood samples were stored in a cooler in ice and water from 1 to 5 hr and then centrifuged in a clinical centrifuge for 10 min. The blood plasma was transferred to two cryovials, frozen at -5C, and later transferred to a -200C freezer. Plasma testosterone and estrogen levels were measured by radioimmunoassay (RIA) (20). The plasma samples were extracted (100 p1l) by adding 5 ml diethyl ether to each sample and freezing the sample on dry ice and acetone. The ether phase was removed and evaporated and then reconstituted with assay buffer. Each sample was analyzed in duplicate. Validation of these assays for snapping turtles was based on the demonstration that serial dilutions of turtle plasma were parallel to the standardized curves (not shown). Chlorinated hydrocarbon analysis. Blood plasma samples were frozen at -20C until preparation for analyses by capillary gas-liquid chromatography. A total of 9 plasma samples from male snapping turtles from Cootes Paradise, 10 from Jack Lake, 4 from Lake Sasajewun, and 2 from Lynde Creek Marsh, was analyzed for PCBs and pesticides. Blood samples from females were pooled for each site and analyzed as single samples for non-ortho PCBs and PCDDs/polychlorinated dibenzofurans (PCDFs). Only samples from females were used because the entire samples from males were used for PCB and pesticide analyses. Six samples from Cootes
Table 1. Comparison of CL, carapace length-adjusted PPR, and precloacal length of male snapping turtles among sites Site CL (cm)* PPR** Precloaca (cm)*** Lake Sasajewan (R) 32.67 3.67 AB 1.324 0.173 A 13.80 2.49 AB (n= 86) Jack Lake (R) 32.67 3.43 AB 1.328 0.121 A 14.74 1.98 A (n= 14) Cootes Paradise (Con) 32.90 4.51 B 1.123 0.141 B 13.30 2.76 B (n= 75) 1.136 0.116 B 12.75 2.46 C 29.86 4.94 A Big Creek (Con) (n = 37) Lynde Creek (Con) 31.83 2.48 AB 1.165 0.146 B 13.83 2.26 ABC

steroid levels and morphology with latitude. Analysis of variance (ANOVA) was used to determine if there were any differences in mean hormone levels or morphology among sites. Because the blood sampling periods among sites did not completely overlap and body size varied among sites, we used carapace length and Julian date as covariates to eliminate variation in hormone levels due to these confounding factors. The arithmetic means and standard deviations (SD) of PCB and pesticide concentrations were not lipidnormalized because the lipid content was so low as to be unmeasurable for most samples. ANOVA was used to find any differences in PCB and pesticide concentrations among sites. Partial correlations were used to measure relationships between testosterone levels and concentration of organochlorine compounds, both within sites and between sites. Because the estrogen levels were highly variable and sample sizes were so low, the partial correlations between estrogen and organochlorine were spurious; thus, we did not present the results here. We used Spearman rank correlations to test for relationships between the pooled PCDDs/PCDFs and non-ortho PCBs from each site and between the mean testosterone and estrogen levels of both male and females for each site. Linear regressions were used to determine if PCB and pesticide concentrations varied with body size. A modified Tukey test for unequal sample sizes was used for post hoc multiple comparisons (27) for all ANOVAs and ANCOVAs. This study complied with the University of Guelph animal care protocol.
Table 2. Comparison of CL, carapace length-adjusted PPR, and precloacal length of female snapping turtles among sites Precloaca (cm)*** CL (cm)* PPR** Site 7.71 1.07 A 0.823 0.09 A 28.13 2.36 AC Lake Sasajewun (R)

(n= 195) Jack Lake (R)

23.76 6.07 ABC

27.00 2.33 AB

0.752 0.076 ABC

0.647 0.108 B

8.16 0.84 ABC

6.74 1.37 B

Cootes Paradise (Con)
(n= 70) Big Creek (Con) (n= 156) Lynde Creek (Con)
25.99 2.74 AB 28.52 1.07 BC

0.633 0.084 C

0.626 0.081 AB

6.26 0.86 C

6.33 0.085 ABC
Abbreviations: CL, carapace length; PPR, precloacal:posterior lobe ratio; R, reference site; Con, contaminated site; SD, standard deviation. Values of carapace length are mean SD, and PPR and precloacal length are least square mean SD. Similar letters (A,B,C) indicate no significant differences among sites (p<0.051. *ANOVA, F= 18.83; p = e.eooi. **ANCOVA, F= 80.86; p<e.ooo1. ***ANCOVA, F= 57.55; p<O.OOOl.
three Great Lakes' sites (contaminated sites) were significantly smaller than those from Jack Lake and Lake Sasajewun (reference sites) (Table 1). There were no significant within-site differences among either the reference sites or contaminated sites. Although there were no significant differences in the PPRs or precloacal lengths between females from Jack Lake and the three contaminated sites, the PPRs and precloacal lengths of females from two of the contaminated sites were significantly smaller than those from Lake Sasajewun (Table 2). Similarly, the precloacal lengths of both sexes were longer at control sites than at most contaminated sites. Only the male PPR was significantly different between Lynde Creek Marsh and the control sites, but since the sample from Lynde Creek Marsh was small, it was difficult to detect a difference. There were dear differences in the modal distribution of PPR in the turtles between males and females from Cootes Paradise and Lake Sasajewun (Fig. 2). Males had larger

Results Morphology. The PPRs of males from the
PPRs than females at both sites. A significantly larger proportion of male snapping turtles from Cootes Paradise had PPRs that overlapped female PPRs than did males from Lake Sasajewun (x2 = 12.50; p = 0.0004; df = 1) (Fig. 3). The proportion of female PPRs that overlapped male PPRs was not significandy different between Lake Sasajewun and Cootes Paradise ( x2 = 3.20, p = 0.0737; df= 1) (Fig. 3). When we compared observer measurements within the same site (Cootes Paradise), the observers' bias in measurement error was of a smaller magnitude than the differences in morphology among sites. Testosterone. Table 3 summarizes the mean testosterone levels at different sites, although two females from Cootes Paradise were excluded as outliers because the estimated testosterone levels were two orders of magnitude larger than the rest. Only at Cootes Paradise were there sufficient observations to examine the relationship between testosterone levels and morphology. Not all observations were used in the following analyses because of occasional missing values from the morphological observations: testosterone levels of males increased linearly with Julian date [adjusted (adj) r2 = 0.2038; F = 7.401; p = 0.0119; n = 26]; carapace length (adj r2 = 0.3034; F= 11.888;p= 0.0021; n = 26); precloacal length (adj r2 = 0.2730; F= 10.390; p = 0.0036; n = 26); and the PPR
(adj r2 = 0. 1997; F= 7.478; p= 0.0113; n= 26). However, carapace length, precloacal length, and PPR all had low tolerances (0.04, 0.02, and 0.1, respectively), and thus were intercorrelated. Precloacal length was eliminated to reduce multicollinearity, and the relationship between the remaining factors and testosterone was assessed by a backward stepwise multiple regression. Testosterone had a significant relationship with both carapace length and Julian date (adj r2 = 0.4957; F= 12.797; p = 0.0002; n = 25). Julian date (adj r2 = 0.0518; F= 2.913; p = 0.0970; n = 36), carapace length (adj r2 = -0.0302; F= 0.0034; p = 0.9503; n = 35), precloacal length (adj r2 = -0.0228; F= 0.3760; p = 0.5449; n = 29), and PPR (adj r2 = -0.0361; F= 0.0240; p = 0.8781; n = 29) did not regress significantly with testosterone in females. Again, predoacal length had a low tolerance (0.08) and was eliminated, and a backward stepwise multiple regression showed no relationship between testosterone and carapace length andJulian date (adj r2 = -0.0355; F= 0.4166; p = 0.6628; n = 35) in females. Carapace length differed among sites in male snapping turtles (Table 1). Because carapace length and Julian date varied linearly with testosterone levels in males, differences in testosterone due to body size and time could be removed using carapace length and Julian date as covariates. There

a a CL

'i to. 30

E 20 E

0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

1.1 1.2 1.3 1.4 1.5 1.6

was a significant difference in mean testosterone levels among sites after adjustment for carapace length and Julian date for males (ANCOVA, F= 5.374; p = 0.0043; n = 32). Testosterone levels of males from Lake Sasajewun were significantly lower than levels of males from Jack Lake, but there were no other differences. Although body size varied among sites for female snapping turtles (Table 2), no covariates were used to compare mean testosterone levels among sites for females because there was no relationship between body size or Julian date with testosterone. There were no significant differences in mean testosterone levels of females (Table 3) among sites (ANOVA, F = 0.3347; p = 0.8003; n = 50). Estrogen. Table 3 summarizes the mean estrogen levels at different sites. Only at Cootes Paradise were there sufficient observations to compare between estrogen levels and morphology. One male was excluded as an oudier, as his estimated estrogen level was an order of magnitude larger than in any other male. Not all observations were used in the following analyses because of occasional missing values from the morphological observations. In our multiple regression analyses, estrogen levels and all independent variables were transformed, using the reciprocal transformation, to normalize the residuals. There was no relationship between estrogen levels of males and Julian date (adj r2 = 0.0005; F= 1.0118; p = 0.3245; n = 26), but there was a positive linear relationship between estrogen and carapace length (adj r2 = 0.1411; F= 5.1076; p = 0.0332; n = 26), precloacal length (adj r2 = 0.1627; F= 5.8566; p = 0.0235; n = 26), and PPR (adj r2 = 0.2372; F= 8.7740; p = 0.0068; n = 26). A backward stepwise multiple regression of estrogen with Julian date, carapace length, and PPR eliminated all variables as insignificant except PPR Predoacal length was removed due to multicollinearity. There was no relationship between estrogen levels of females and Julian date

0.5 0.8

Fe:t-40 ~4
Cootes Paradise Lake Sasajewun

1.6 1.7

PPR Figure 2. Frequency histogram of precloacal:posterior lobe ratios (PPRs) of snapping turtles at Cootes Paradise in 1994-1995 (males, n 111; females, n 125) and at Lake Sasajewun in 1987-1991 (males, n 39; females, n 1).

= = = =

Figure 3. Proportion of male and female precloacal:posterior lobe ratios (PPRs) that overlap with the PPRs of the other sex at Cootes Paradise and Lake

Sasajewun.

Volume 106, Number 5, May 1998
Environmental Health Perspectives
(adj r2 = -0.0263; F= 0.3855; p = 0.5408;
n = 32), carapace length (adj r = -0.0298; F= 0.1318; p = 0.7192; n = 31), precloacal length (adj r2 = -0.0393; F = 0.0917; p = 0.7641; n = 25), or PPR (adj r2 = -0.0404; F= 0.0691; p = 0.7950; n = 25). A backward stepwise multiple regression of estrogen with Julian date, carapace length, and PPR eliminated all variables as insignificant. Precloacal length was removed due to multicollinearity. Because carapace length and Julian date varied linearly with estrogen for males, carapace length and Julian date were used as covariates to compare mean estrogen levels among sites. There was no difference among sites in estrogen levels of males (ANCOVA, F= 0.8629; p = 0.4331; n = 35). No covariates were used to compare mean estrogen levels among sites for females because estrogen levels were independent of Julian date and carapace length. There was no difference in mean estrogen levels among sites for females (ANOVA, F = 0.4245; p = 0.7364; n = 48). There was a significant difference in testosterone levels between males and females at Cootes Paradise, where carapace length and Julian date were covariates (ANCOVA, F= 64.0372; p<O.0001; n = 60), but not estrogen (ANCOVA, F = 0.7440; p = 0.3923; n = 57). Latitude. There was no relationship between levels of testosterone and latitude in either males (adj r2 = -0.0254; F= 0.0728; p = 0.7888; n = 38) or females (adj r2 = 0.0024; F= 1.102; p = 0.2977; n = 47). We transformed the estrogen levels and latitude using the reciprocal transformation prior to regression analysis. There was no relationship between estrogen and latitude for either males (adj r2 = -0.0182; F= 0.3550; p = 0.555 1; n = 37) or females (adj r2 = -0.0222; F= 0.0248; p = 0.8756; n = 47). In males, there was a significant relationship between latitude and both size-adjusted precloacal length (adj r2 = 0.0968; F= 23.943; p<0.0001; n = 215) and PPR (adj r2 = 0.1515; F= 39.213; p<O.0001; n = 215). Similarly, in females, there was a significant relationship between latitude and size-adjusted precloacal length (adj r2 = 0.0968; F= 23.943; p<0.0001; n = 461) and PPR (adj r2 = 0.1515; F= 39.213; p<O.0001; n = 461). These regressions were subdivided into contaminated and control sites, but there were no dear trends of either size-adjusted precloacal length or PPR with latitude within these two categories of sites. Chlorinated hydrocarbons. PCB and pesticide levels were higher in blood plasma of males from Cootes Paradise and Lynde Creek than of males from Jack Lake or Lake Sasajewun (Table 4). There were few

differences between either Cootes Paradise and Lynde Creek or between Jack Lake and Lake Sasajewun except for p,p'-DDE and total PCB concentrations. However, we had low power to detect any differences due to our small sample sizes. Because there were significant relationships between testosterone concentrations and both carapace length and Julian date, a partial correlation was performed to estimate the effect that PCB and pesticide concentrations have upon testosterone levels at each site. After partialling out the effects of body size and Julian date, there were no significant relationships between testosterone levels and either PCB or pesticide concentrations at either Cootes Paradise or Jack Lake (Table 5). The partial correlations were almost equally divided between positive and negative values. Similarly, there were no significant relationships between mean testosterone levels and either mean PCB or mean pesticide concentrations among all sites, although most of the values were positive.
Although 70.6% of the PCDDs/ PCDFs and non-ortho PCBs detected in the pooled samples were higher in blood plasma collected at Jack Lake than at Cootes Paradise and 57.9% were higher at Jack Lake than at Lynde Creek Marsh, the concentrations were of the same magnitude (Table 6). If the concentrations were higher at either Cootes Paradise or Lynde Creek than at Jack Lake, then the concentrations differed by at least an order of magnitude. Generally, the contaminant levels were highest and similar in Cootes Paradise and Lynde Creek Marsh, lower in Jack Lake, and lowest in Lake Sasajewun. Similarly, there were no significant relationships between the pooled PCDDs/PCDFs and non-ortho PCBs from each site with the mean testosterone levels of either males or females.

Discussion

Our hypothesis that the sexually dimorphic morphology of adult snapping turtles would be more feminized at contaminated sites was
Table 3. Mean concentrations SD of testosterone and estrogen in blood plasma (ng/ml) of snapping turtles from Cootes Paradise, Jack Lake, Lake Sasajewun, and Lynde Creek Marsh in 1995 Male

Articles * Organochlorine contaminants in snapin turtles
(30,33). We did not find any apparent peak in any of the hormone profiles; instead, the testosterone levels kept increasing until August, when sampling stopped. Other studies have found that estrogen levels in snapping turtles are lowest in June during ovulation and the postovulation phase, and increase again in the fall during vitellogenesis (31,32). However, we found that mean estrogen levels were constant over time, probably because we caught female snapping turtles mostly during the ovulatory and postovulatory period. There was no evidence that contaminants had any effect upon testosterone or estrogen levels. Although many partial correlations were negative, there were no significant relationships between testosterone levels and any of the PCBs and pesticides within either Cootes Paradise or Jack Lake. Similarly, there was no relationship between testosterone or estrogen and either PCBs or pesticides among sites. Surprisingly, although the relationships were all nonsignificant, most were positive, which is the opposite of what was predicted. As expected, the concentration of most of the PCBs and pesticides measured were lower in turtles from Jack Lake or Lake Sasajewun than in those from Cootes Paradise or Lynde Creek. Nevertheless, 12 of the 16 PCBs and pesticides tested for were found, albeit at low concentrations, in the samples from the control sites. We did not find the same trend with PCDDs/PCDFs and non-ortho PCBs. Although the pooled levels of PCDDs/ PCDFs and non-ortho PCBs were lowest at Lake Sasajewun, there were no differences among Jack Lake, Cootes Paradise, or Lynde Creek, and for many of these contaminants, the levels were highest at Jack Lake. These results are likely confounded by pooling samples; thus, we could not adjust our analyses with covariation with body size. There were significant regressions between carapace length and PCB or pesticide concentrations in 8 of the 17 compounds examined from the Cootes Paradise turtles, and every regression but one (nonsignificant) was positive. Conversely, none of the 13 regressions between carapace length and PCB or pesticide concentrations from Jack Lake was significant. Bishop et al. (34) found no relationship between female body size and organochlorine contaminant concentrations in snapping turtle eggs. However, there was a positive relationship between body size and organochlorine concentration in body tissues (23,35). We found that larger turtles had higher concentrations of PCBs and pesticides in blood plasma, at least when the level of contamination was already high. This suggests that the trends in organochlorine contamination in blood plasma follow

the same trends as do other tissues. Because turtles from Jack Lake did not show a relationship between body size and contaminant concentration, either we did not have sufficient power to detect the relationship or the contaminants were being metabolized or removed at a rate equal to the rate of uptake. A power analysis (36) with a medium effect size off2 estimated 1- ,B = 0.1911 [TJ,8) critical = 5.3177; X = 1.5] for the regressions between carapace length and PCB and pesticide concentrations. Therefore the nonnegative regressions were inconclusive (32), as we did not have enough power to detect a difference unless the effect size was very large. Our two main results seem contradictory. There is no evidence that testosterone production has been impaired or estrogen production enhanced in snapping turtles with high levels of organochlorine contaminants. However, there is evidence that there is some feminization occurring at the contaminated sites. p,p'-DDE inhibits sexual development in rats without affecting plasma testosterone levels (38). The concentration ofp,p'-DDE that could inhibit androgen receptor activity in the rat was 63.6 ng/g ww (38), which is of the same magnitude as the mean concentration of p,p'DDE in the plasma of adult Cootes Paradise turtles (10.1 ng/g ww), total DDT in snapping turtle muscle tissue from the Niagara peninsula in 1989 (164.60 ng/g ww) (23), and p,p'-DDE in snapping turtle eggs from Cootes Paradise in 1991 (5,910 ng/g ww) (4). Previous studies have shown that p,p'-DDE is an androgen receptor antagonist and raises the possibility that the changes in sexual characteristics could relate to antiandrogenic activity. These concentrations of p,p'-DDE are sufficiently high to inhibit androgen receptor transcription ability in vitro in rats (38). Similarly, PCBs have been shown to induce changes in sexual development without alterations to testosterone levels (39). Our study suggests that external sexual development may be more sensitive to exposure to environmental contaminants than testosterone production or metabolism of adults. Continued exposure during sexual development of juveniles may also contribute to alterations in sexually dimorphic morphology, particularly since the dose of the exposure increases as the turtle gets larger. Biomonitoring often involves either sampling body tissue (2,5,23) or eggs (4,5,21,22). Unfortunately, although snapping turtles make good environmental indicators, snapping turtle populations are highly susceptible to increased adult mortality (40,41). Repeated or large-scale lethal tissue sampling of snapping turtle adults is undesirable, particularly if the population is already in decline. Although

14. Facemire CF, Gross TS, Guillette W Jr. Reproductive impairment in the Florida panther: nature or nurture? Environ Health Perspect 103(suppl 4):796 (1995). 15. Munkittrick KR, Portt CB, Van Der Kraak GJ, Smith IR, Rokosh DA. Impact of bleached kraft mill effluent on population characteristics, liver MFO activity, and serum steroid levels of a Lake Superior white sucker (Catostomus commerson,) population. Can J Fish Aqua Sci 48:1371-1380 (1991). 16. Fry DM, Toone CK. DDT-induced feminization of gull embryos. Science 231:919-924(1981). 17. Gibbons W, Lovich JE. Sexual dimorphism in turtles with emphasis on the slider turtle (Trachemys scripta). Herpetol Monogr 4:1-29 (1990). 18. MacDonald CR, Metcalfe CD. Concentration and distribution of PCB congeners in isolated Ontario lakes contaminated by atmospheric deposition. Can J Fish Aquat Sci 48:371-381 (1991). 19. Mosimann JE, Bider JR. Variation, sexual dimorphism, and maturity in a Quebec population of the common snapping turtle, Chelydra serpentina. Can J Zool 38:19-38 (1960). 20. McMaster ME, Munkittrick MR, Van Der Kraak GJ. Protocol for measuring circulating levels of gonadal sex steroids in fish. Can Tech Rep Fish Aquat Sci 1836
biphenyls (PCBs) by glass capillary chromatography. Fresenius Z Anal Chem 302:20-31 (1980). 25. Norstrum RJ, Simon M. Determination of specific polychlorinated dibenzo-p-dioxins and dibenzofurans in biological matrices by gel-permeation/carbon chromatography and gas chromatography-mass spectometry. In: Environmental Carcinogens, Methods of Analysis and Exposure Measurement, Vol 11. Polychlorinated Dibenzo-p-dioxins and Dibenzofurans (Rappe C, Buser HR, Doget B, O'Neill IK, eds). Lyon:international Association for Research on Cancer, 1991;281-297 (1991). 26. StatSoft, Inc. Statistica for Windows. General Conventions and Statistics I. Tulsa, OK:StatSoft, Inc., 1995. 27. Spotvoll E, Stoline MR. An extension of the T-method of multiple comparisons to include the cases with unequal sample sizes. J Am Stat Assoc 69:975-978
28. Munkittrick KR, Van Der Kraak, Washington GJ, McMaster ME, Portt CB. Response of hepatic MFO activity and plasma sex steroids to secondary treatment of bleached kraft mill effluent and mill shutdown. Environ Toxicol Chem 11:1427-1439 (1992). 29. Ernst CH, Barbour RW, Lovich JE, eds. Turtles of the United States and Canada. Washington, DC:Smithsonian Institution Press, 1994. 30. Mahmoud IY, Cyrus RV, Bennett TM, Woller MJ, Montag DM. Ultrastructural changes in testes of the snapping turtle, Chelydra serpentina, in relation to plasma testosterone, A5-3p-hydroxysteroid dehydrogenase, and cholesterol. Gen Comp Endocrinol 57:454-464 (1985). 31. Lewis J, Mahmoud IY, Klicka J. Seasonal fluctuations in the plasma concentrations of progesterone and oestradiol-17, in the female snapping turtle (Chelydra serpentina). J Endocrinol 80:127-131 (1979). 32. Mahmoud IY, Cyrus RV, Woller MJ, Bieber A. Development of the ovarian follicles in relation to changes in plasma parameters and 5A3P HSD in

ping turtles (Chelydra serpentina). Can J Zool 74:291-296 (1996).
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