Science in the box

The tiered-effect process progressively takes more complex approaches to ecotoxicological assessments as the data and testing becomes more detailed and refined.

The goal of ecotoxicology is to understand the concentration of chemicals at which organisms in the environment will be affected. This exposure concentration should be avoided in order to protect the environment.

To study the possibility that a chemical is toxic, ecotoxicologists usually start with simple, conservative approaches. They progress to more realistic but also more complex approaches only when more accurate information is needed. This process is referred to as the "tiered-effect process."
As scientists move through the tiers, ecotoxicity data becomes more relevant, but it also takes more time and resources to obtain the data.

Currently, the effects assessment approach starts with a QSAR approach and progresses to Acute, Chronic, and Model Ecosystem (mesocosm) testing as needed. Ecotoxicity data is combined with fate data in order to reach a decision about whether the ingredients are safe to use in products. These decisions are made in the environmental risk assessment.

Even after we are done understanding the ecotoxicity and conducting the risk assessment, the work is not always finished. We may continue to work to understand the environment and how the chemistry and physical nature of the environment interact to affect the health of the community. This research area is called Eco-epidemiology.

Why In Vitro?
In vitro testing uses cells and tissues outside of the body to test for environmental-health risks. As the accuracy of these tests improves, there is less demand to do ecotoxicity tests on living beings.
In human safety studies, in vitro methods (using cells and tissues outside the body in an artificial environment) are routinely used to determine the safety or effectiveness of a drug or an ingredient in cleaning products. In environmental testing programmes, we are trying to use similar in vitro methods to study the toxicity of chemicals to fish.
The plan is that these approaches will one day allow us to greatly reduce or eliminate the need for fish in our work. The problem is that, at this stage, we do not know enough about how well in vitro tests predict the response of whole fish to chemicals.

Methods
In vitro testing appears simple. Cells from the body of a living organism are grown with a chemical soup that provides the nutrients needed for the cells to survive outside the body. These cells are exposed to ingredients, and each ingredient is analysed to understand its metabolism. The cells are analysed to understand the concentration of the ingredient that makes the cells unhealthy.
In fact, this is highly challenging, primarily due to the fact that it is very difficult to keep cells healthy outside of the body. As our knowledge of biology grows, we will apply this information to in vitro biology and in vitro testing.

In Vitro Testing
In vitro testing can provide means to determine the ingredient concentration level at which plants and animals may become at risk (without animal testing). Although there are many challenges, there has been some success.

Our Goals
The goal of effects testing is to find the concentration of an ingredient that adversely affects plants and animals in the environment. We do this to make sure our ingredients are well below that concentration in the environment. To avoid the use of animals, we are working on developing in vitro methods. Since organisms are made up of cells, we use cells in effects tests and try to find the concentration that affects cells. In the assessments, this concentration is then assumed to be the same as the concentration that affects the whole organism. This research has been ongoing in human health with some initial success.

Challenges
It is a challenge to use just the cells of one organ to tell us how another organ of the body would respond to a chemical. Furthermore, living organisms are very complex and only function when all the cells of the body are working together. Finally, some organs, like the liver, can take a chemical and change its toxicity or can remove the chemical from the body. These interactions among organs make it difficult to develop one or a few in vitro techniques to provide effects data for one species of fish, much less to provide toxicity data on multiple species of fish and invertebrates. While this task is complex and difficult, the industry is working to develop these methods and thereby reduce animal testing.

The Bioconcentration Factor (BCF)
Bioconcentration testing measures how much of a substance can be found in an organism compared to its presence in the environment (eg, how much of a surfactant is in a fish compared to the concentration in the water). The K_ow (octanol-water partition coefficient) has many variable factors, including a chemical’s solubility and an organism’s metabolism.
Animals living in the aquatic environment take up chemicals from water as well as from the food they eat. For many chemicals, the concentration in the animals is higher than the concentration in the fish.

Scientists measure the chemical's bioconcentration factor (or BCF), which tells us how much more of the chemical is in the fish than in the water. If the BCF=2, the concentration of the chemical in the organism will be twice the concentration in the water. This is important because the amount of a chemical that gets into an animal helps to determine the toxicity of that compound. The bioconcentration factor (BCF) also helps scientists to calculate how much of a chemical an organism will take up from its food.
BCF can be measured in the laboratory or calculated using computer models (see QSARs). When we measured surfactant BCF values in fish, we noticed they were lower than predicted by computer models. These models were based on a physical chemical property called the octanol-water partition coefficient (or Kow). The Kow measures how a chemical partitions between water and a fat substitute (octanol), which basically tells the scientist how soluble the chemical is in fat, since the fat is where many chemicals get stored in the body.

Metabolism
Unfortunately, these computer models do not consider the possibility that chemicals can be metabolized by the organism. Metabolism is the breakdown of food and other chemicals into smaller molecules. These smaller molecules are typically quickly eliminated from the body. We think the reason the measured bioconcentration factor values were lower than the computer model predicted was due to metabolism in fish. To help figure this out, P&G uses an in vitro system to study the metabolism of chemicals in fish.

In Vivo Data
In vitro studies have been conducted on selected chemicals (particularly surfactants) for which data was compared to in vivo data. This research helped to develop toxicity, BCF and metabolism test methods that do not use animals. This work is linked with our work on QSARs, and we hope to further improve these tools so that we won't have to conduct even in vitro work in the future.

Acute toxicity tests assess at which level of exposure the growth or survival of certain indicator organisms will be affected. Different organisms react differently so it is important to test plants, invertebrates and fish to determine which substances may be toxic to which organisms at which levels.

Short-Term Testing
Acute toxicity or acute effects tests are rapid procedures (2 to 4 days) used to measure the concentration that will negatively affect the test organisms.
Data from these tests can be used to:

• screen for toxicity (determine if the compound is toxic)
• rank toxicity to identify the best ingredients to continue investigating for use in a product, and,
• assess the potential for effects in the environment.

In some cases, one group of organisms will be more sensitive to a compound than another group. For example, insecticides are usually more toxic to invertebrates than to fish or algae.
When we start a toxicity test programme, we may not know which group will be most sensitive to the new compound. So we usually test at least one plant, one invertebrate, and one fish species. It is important that all three groups of organisms are tested because all are important in the environment, and effects on a plant may not tell us anything about effects on an animal and vice versa.

Lethality (mortality) is the most common endpoint for invertebrates and fish, while growth of a population of cells is used to understand effects on algae. Of course, aquatic ecosystems are composed of hundreds, or even thousands of different species.
The process we use to protect all these different species is called environmental risk assessment . When acute toxicity data does not provide enough information for us to decide if the compound is safe or not, we conduct chronic toxicity tests.

Chronic toxicity tests examine any long-term effects a substance may have on an organism’s growth, reproduction and survival. Such chronic toxicity exposure tests start typically on very young organisms through reproduction and measured against the health of non-exposed control organisms.

Conducting Chronic Toxicity Tests
In chronic and acute tests, we expose the test organism to the substances found in P&G’s cleaning products, for a substantial portion of its lifetime and look for effects on growth, reproduction and survival.

Testing through the Entire Life
Typically, young organisms are more sensitive to chemicals than older organisms. So, we usually start tests with very young organisms and expose them to a chemical for as long a period of time as is practical. Since reproduction is generally a sensitive endpoint, tests are often continued until reproduction begins. Many species produce several offspring every few days, and during a chronic toxicity test we get dozens to hundreds of young produced. In all of these tests, the health of the treated organisms is compared with that of control organisms. Except for the presence of the chemical, both the treated and control organisms are treated under the same conditions. As long as the exposed organisms can survive, grow and reproduce as well as or better than the control group, we know our chemical is not harming them. The lowest tested concentration without effect on the test chemical is called the "No Observed Effect Concentration" (or NOEC).

Conditions
We will test one to fifteen species in chronic toxicity tests with our chemicals. These species are tested one at a time in the laboratory under carefully controlled conditions. These conditions help to keep the organisms healthy and increase their growth rate and reproduction. In the environment, organisms do not live alone with all of their needs (food and water) provided. Instead, organisms depend on each other to provide food and shelter in a food-web or a community of organisms. The complex interactions that can occur in a community cannot be studied by isolating organisms and testing each organism separately in the laboratory. To make sure we are not affecting the interactions among species, we sometimes conduct tests with many organisms at the same time. This research is called model ecosystem research.

Because of the complex nature of organisms in the environment, P&G set up model ecosystems or mesocosms to test the effects of substances on organisms within as close to natural conditions as possible.

The goal of all the environmental fate and effects testing at P&G is to make sure our products and their ingredients will not affect the environment. So, the best place to test the toxicity of our ingredients would be in the real environment. But, because of the value of the environment and the possibility of negative effects from our testing, this would not be an appropriate approach. Instead of using the real environment, we use small parts of the environment that have been isolated. These are called "model ecosystems" or"mesocosms".
A model ecosystem is the highest tier of ecotoxicity testing available. Definitions of model ecosystems vary, but they are usually fairly large (>10 meters long for streams, >10,000 litres for ponds), can be indoors or outdoors, contain a diverse array of species and are sustainable for long periods of time. Basically, these are slices of the environment that have been moved into the laboratory for experimental work.
The advantages for using model ecosystems or mesocosms are many and include:

• They allow study of environmental fate and effects at the same time.
• The test system is very realistic and helps us understand how to use in vitro, acute and chronic information to better protect the environment
• Testing is carefully controlled (light levels, chemical concentration, river flow). This level of control could not be found in the environment.
• Complex aquatic communities containing hundreds of species can be evaluated at the same time. Most of these species cannot be cultured or tested under laboratory conditions.
• Interactions among species are included so that effects on one species can cause effects on other organisms.

P&G developed its Experimental Stream Facility or mesocosm in the mid 1980s, generating important data for 15 years before donating it to a local university.

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