TOXICITY 101

What is Ecotoxicity

A science that studies the combination of ecology and toxicology. This science refers to the potential for biological, chemical or physical stressors to affect ecosystems. Ecotoxicology has been defined as, “the branch of toxicology concerned with the study of toxic effects, caused by natural or synthetic pollutants, to the constituents of ecosystems, animal (including human), vegetable and microbial, in an integral context”.


Ecotoxicity testing refers both to the assessment of chemical effects on fish, birds, or other wild organisms and the testing of soil, sediment, or effluents for the presence of toxic compounds.

What is toxicity

Toxicity is the degree to which a chemical substance or a particular mixture of substances can damage an organism. Toxicity can refer to the effect on a whole organism, such as an animal, bacterium, or plant, as well as the effect on a substructure of the organism, such as a cell (cytotoxicity) or an organ such as the liver (hepatotoxicity). 
The toxicity of a substance is not an inherent property but the detrimental manifestation of its biochemical effect in a living system. The acute toxicity of a substance is measured by the amount needed to kill half the population of a test species, called the EC50 (effective concentration 50%)

Aqua Science main focus is providing a reliable way to test for acute toxicity. The BioLight Toxyluminometer along with the BioLight line of custom reagents and consumables offers this for both the field and the bench top. The BioLight Reagent is based on a marine bacterium, AliiVibrio Fishceri which elicits light. This well-known method of detecting acute toxicity offers an accurate and repeatable test.

History of Toxicity

“Toxic” and similar words derive from Greek τοξον toxon (“bow”), a reference to the use of poisoned arrows as weapons. This root was chosen because the transliteration of ‘ιον ion, the usual Classical Greek word for “poison”, was not distinctive enough from the English word “ion,” itself derived from a similar but unrelated Greek root.

scientists in lab

Types of toxicity

acute toxicity

Acute Toxicity

Acute toxicity describes the adverse effects of a substance that result from single exposure. Acute toxicity looks at lethal effects following oral, dermal or inhalation exposure.


An acute toxicity refers to a poisonous state (and its adverse effects) that has a combination of the following aspects:

All chemicals, drugs, and natural substances are potentially poisonous, or toxic, at high enough doses. Acute systemic toxicity tests identify short-term toxic effects that appear soon after a substance is swallowed (oral toxicity tests), absorbed through the skin (dermal toxicity tests), or inhaled (inhalation toxicity tests). If appropriate, data from acute systemic toxicity tests are used to develop warning labels, protective packaging, occupational personal protective equipment requirements, and environmental release limits.

Chronic Toxicity

Chronic toxicity describes the adverse effects of a substance from long-term exposure. Chronic toxicity, the development of adverse effects as a result of long term exposure to a contaminant or other stressor, is an important aspect of aquatic toxicology. Adverse effects associated with chronic toxicity can be directly lethal but are more commonly sublethal, including changes in growth, reproduction, or behavior. Chronic toxicity is in contrast to acute toxicity, which occurs over a shorter period of time to higher concentrations. Various toxicity tests can be performed to assess the chronic toxicity of different contaminants, and usually last at least 10% of an organism’s lifespan. Results of aquatic chronic toxicity tests can be used to determine water quality guidelines and regulations for protection of aquatic organisms.

chronic toxicity
compliments analytical testing

How Toxicity Testing complements traditional chemical analytical testing?

Water quality assessment and characterization techniques typically rely on analyses and data for individual contaminants. Essentially, individual chemical concentrations in water are measured or modeled.
These results are then compared to the known concentrations at which the chemicals have been shown to cause adverse health outcomes (usually mortality, growth, or fecundity). This approach is helpful to approximate the risk posed by known chemicals; however, it may have limited usefulness.

Real-world exposures generally do not occur from individual chemicals; they typically occur as mixtures of different chemical compounds that can change over time. This presents challenges in understanding the risks posed to human and environmental health: (1) many chemicals lack toxicity data even if measurable, which means there is a lack of information on the effects to exposed organisms; (2) although individual compounds can accurately be measured at low concentrations, information is lacking on the presence and concentrations of other compounds; and (3) chemicals in a mixture tend not to work alone, but together additively, synergistically or antagonistically.

To better understand the risk posed by these complex samples, we need methods that can characterize potential cumulative effects on the organisms without necessarily needing to know all the components of the samples.

What is Chemical Toxicity Testing

These are the steps to go through to determine if a substance is toxic and at what concentration levels. In reality, today, toxicity testing is even more complicated and detailed. There are now many measures of toxicity other than death or sickness: for example, many tests done today look at “endpoints” such as effects on enzyme systems, or changes in animal behavior or decreases in egg production. The final use of toxicity data is comparison with concentrations measured or expected in the field. If the concentrations of a pollutant in the field are below any of the concentrations deemed “toxic” in the laboratory, it may well be that the pollutant is not a problem. If concentrations in the field are higher, then there is cause for concern. 

Bioassay

The basic tool for determining toxicity of substances to marine and aquatic organisms is the toxicity test. In its simplest form, toxicity testing is taking healthy organisms from a container of clean water and placing into one containing the same water with a known concentration of a pollutant. The observer then watches to see if, and when, it appears to become lethargic, sick or dies, and comparing those results to the organisms left in the clean water.

lab collaboration
drinking and wastewater testing

Complexities of toxicity testing

The testing process for determining toxicity in marine environments is detailed, rigorous, and time consuming. There must be containers of both the uncontaminated (clean) water (called a control) and the pollutant-treated water; a bare minimum is five containers of each. The reason for the replications is the concept of variability. Given five test organisms, such as a fish species, there will be a range of sensitivity among them. Having multiple testing samples allows scientists to determine the level toxic to the average organism and the level toxic to the most sensitive organism. Having more than one of the same organism in each test container is required; ten is standard. It’s easy to see how a toxicity test grows in complexity: 50 specimens for the controls (10 in each of five replicate containers) and 50 more in the five treated containers (10 in each of five replicate treatment containers). That’s 100 organisms. But then, to find out what concentrations of the toxicant are safe and which are not, there needs to be at least five different treatment concentrations, each with five containers and each container with 10 test organisms. Now we’re dealing with 600 test organisms and 60 test containers.

Observations over time

The next step in a toxicity test is recording the changes in the organisms over time. A standard observation period is daily, every 24 hours for at least 4 days (96 hours). For each interval of time, observations must be recorded for:

 

  • Each of the treatment and control containers
  • The numbers of organisms that are alive and normal
  • The number not doing well
    The number dead

Then apply a statistical procedure to estimate the median concentration of the toxin that maimed or killed half the organisms and write up the results. The key is to write it up with enough information so that someone else can exactly duplicate the test.

observations over time image 2

Rapid Acute Toxicity Using Bioluminescence

Microbial bioassays based on bacterial methods have been conducted for toxicity since the 90s. The application of luminescent bacteria proved to be a useful tool for the assessment and monitoring of toxicity because they are non-invasive, rapid, reproducible and user-friendly. Bioluminescence is closely associated to cellular metabolism, and thus it is a sensitive indicator of metabolic status of the bacteria. In the presence of noxious substances, the cellular activity is inhibited and the light intensity is reduced consequently. The bioluminescence inhibition can be calculated by measuring the light intensity of the sample exposed to a stressor and comparing that with a corresponding control sample. The most important index for toxicity expression in bioassay investigations is the effective concentration (EC50), which is the concentration of a toxic substance that decreases light emission by 50%.

Aliivibrio fischeri (formerly known as Vibrio fischeri) is a marine, Gram-negative, facultative anaerobic and non-pathogenic bacterium. This bacterium is distributed throughout the world preferentially in temperate and subtropical waters existing as as light-producing organs symbionts in some species of squids and fishes. This makes Aliivibrio fischeri the perfect option for use in toxicity monitoring of the water environment.

Aqua Science produces BioLight bioluminescent bacteria (reagent) used for acute toxicity testing. It is freeze dried for extended life then stored frozen at -25 C and is reconstituted before use. A single vial of reagent contains approximately one hundred million cells. The sample is salinity adjusted to ensure a saltwater atmosphere due to Aliivibrio fischeri being a marine bacterium. Diluent is used for dilutions as required for the test protocol. The bacteria elicits the brightest light levels following reconstitution. These light levels are measured prior to sample exposure and over time after exposure to a sample, using a luminometer. The luminometer measures the changes in the light to indicate the levels of toxicity. BioLight Reagents are produced and tested under strict Quality Control and will include a Quality Certificate as per requirements of the International Standard Organization, ISO 11348-3.

 

drinking water testing

TOXkits Using Multi Species

These rapid stock culture-independent toxicity test kits allow industries, water facilities, commercial testing labs and universities to do toxicity testing using multiple different species with results in as little as 2 days following preparation and incubation time. The cultures are dormant or immobilized and ready to use once activated, avoiding the requirement for large tanks and healthy species as used in bioassay testing. The test species and the test protocols are the same as in many standardized toxicity tests conform with ISO Standards, OECD Guideline or EPA guidelines. There are optional tests and test species for fresh water, wastewater, seawater, estuaries, sediments, soils and solid wastes. The TOXKITS offer a whole battery of ecotoxicological bioassays with crustaceans, micro-algae, protozoa, rotifers and higher plants.

Options for TOXkits

Sheepshead minnow, Cyprinodon variegatus
Silverside, Menidia beryllina, Menidia menidia, and Menidia peninsulae
Mysid, Americamysis bahia (formerly Mysidopsis bahia

Some Chronic Toxicity – Marine and Estuarine

Sheepshead minnow, Cyprinodon variegatus, larval survival and growth
Sheepshead minnow, Cyprinodon variegatus, embryo-larval survival and teratogenicity
Inland silverside, Menidia beryllina, larval survival and growth
Mysid, Americamysis (formerly Mysidopsis) bahia, survival, growth and fecundity
Sea urchin, Arbacia punctulata, fertilization

Fathead Minnow, Pimephales promela, and Bannerfin shiner, Cyprinella leedsi
Daphnia, Ceriodaphnia dubia
Rainbow trout, Oncorhynchus mykiss, and Brook trout, Salvelinus fontinalis
Daphnia puplex and Daphnia manga

 

Some Chronic Toxicity to Freshwater Organisms

Fathead minnow, Pimephales promelas, larval survival and growth
Fathead minnow, Pimephales promelas, larval survival and teratogenicity
Daphnia, Ceriodaphnia dubia, survival and reproduction
Green alga, Selenastrum capricornutum, growth (species is now known as Raphidocelis subcapitata)

Why test for toxicity?