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Direct Toxicity Tests


Acute and Chronic Direct Toxicity Tests

Biological assays to detect toxicity  

direct toxicity tests DaphniaThe Direct Toxicity Tests utilized at EBPI Analytics offer acute, sub-chronic and chronic endpoints for a variety of bacteria, micro fauna, marine invertebrates, vertebrates and plant species. The data collected from these assays provide LC50, LOEL and NOEL concentration data for test compounds in aqueous samples (fresh and marine water) and sediment samples.

This data is required in many areas including:

  • Primary toxicological assessments
  • Drinking/waste water testing
  • Pesticide approval     
  • Environmental remediation
  • Pharmaceutical development

direct toxicity tests labplants testtubesA number of these assays are required in international water effluent quality testing protocols including WSER, NPDES, and WFD. The direct toxicity assays run at EBPI Analytics have been used extensively in government, academia, and industry.

Most of the assays provided at EBPI Analytics follow developed standardized protocols in accordance with the ISO, OECD, and EPA. Several of the assays with no current international standard are in the approval process to be standardized.


Direct Toxicity Assay Options

Bioluminescence Inhibition Assay

direct toxicity bioluminescence inhibition assay vibrio fischeriThe bioluminescence inhibition assay uses Vibrio fischeri bacteria to measure acute toxicity of complex effluents, chemcial mixtures and pure compounds. This bioassay has been standardized and is utilized in several countries. Assay bacteria are subjected to the sample mixture and bioluminescence is measured after an exposure time with a luminometer. Inhibition of bioluminence, which is controlled by several bacterial systems and can be affected by protein synthesis, metabolism or energy production, is the response indicator of toxicity. The samples are compared to negative and positive control solutions to obtain data.

Benefits to this assay are

  • Standardized test method run according to ISO 11348-3
  • Relatively quick turn-around time
  • Cost effective
  • Ideal for marine samples


TOXI-CHROMOTEST (2 h, Direct bacterial toxicity assay)

direct toxicity Toxi ChromoTest Platesmalldirect toxicity toxi ChromoTest logoThis assay uses a modified E. coli bacteria to measure direct toxicity though a colorimetric endpoint. Healthy bacteria produce significant amounts of B-galactosidase which is reacted with an added chromogen to produce a colour change. Bacteria that show toxicity produce less of the enzyme and the colour changes are not as dramatic for these samples. The degree of inhibition can be calculated spectrophotometrically, providing a clear, interpretable endpoint for toxicity quantifiation. These assays have been run as a battery of tests used in environmental screening processes and throughout academic research. In 2006, the Toxi-ChromoTest was verified by the ETV program under the U.S. Environmental Protection Agency (EPA).

  • Cost and time effective (1 day for results)
  • A wide range of compounds can be screened for initial toxicity levels
  • Quantification of toxicity allows calculation of threshold values for compounds tested


PROTOXKIT F (24 h, Chronic Protozoan toxicity test, fresh water)

Protozoan toxicity testCiliates are a very important component of benthic micro fauna communities and play a key role in recycling organic matter. They are also, next to bacteria, the second most important group of biota in the activated sludge of waste water treatment plants.This assay is based on measuring optical density (OD) in a solution of ciliate biomass as food substrates are added and subsequently turned over. Inhibited culture growth under toxic stress is indicated by prolonged turbidity of the exposure groups as the food remains in suspension and produces a higher OD in comparison to the control. Mean growth inhibition after 24 h exposure at 30°C is determined and expressed as a 24 h EC50. NOEC and LOEC values can be calculated with specific data treatment program. As the endpoints are non-lethal, chronic effect concentrations can be calculated.

  • Tests are time and cost-effective
  • Sensitive assay that compares to other ciliates in activated sludge, useful for influents of wastewater treatment
  • Results compare well with human and animal cell lines, indicating increased biological relevance
  • NOECs calculated serve as useful thresholds for protozoans in the environment


ROTOKIT F (24 h, Acute Rotifer toxicity test, fresh water)

ROTOKIT M (24h, Acute Rotifer toxicity test, salt water)

direct toxicity protozoanRotifers are important members of many aquatic communities. With copepod and cladoceran crustaceans they are the major constituents of freshwater zooplankton, with higher turnover rates compared to crustaceans. The test organisms are hatched from dormant eggs and exposed to the samples. When a suspension of red microspheres is added, healthy larvae will ingest the microspheres and give a coloured digestive tract that is observable under a microscope. The acute ROTOXKIT assays are 24 h tests based on mortality of the test organisms. Lethal concentrations of test compounds are measured by calculation of the 24 h LC50. The options of the assays offered allow analysis of both salt and fresh water samples.

  • Cost and time effective
  • Measures direct environmental effects on important strata of organisms
  • Sensitive assay
  • The acute test is an ASTM Standard protocol since 1991


ROTOXKIT F (48 h, Chronic Rotifer toxicity test, fresh water)

The method is the similar to the acute Rotifer tests, however the Rotifers in this assay are not given the microbeads and are subjected to conditions that encourage population growth. The endpoint measures the decrease in reproduction of the rotifers under toxic stress after 48 h exposure, with calculation of the 48 h median growth inhibition (48 h EC50). NOEC and LOEC values can be determined with specific data treatment programs.

  • Tests are of high quality and are very reproducible
  • Sensitive assays
  • NOECs calculated through the assays are meaningful thresholds for the impact of tested compounds on this group of aquatic biota.
  • This assay is used in the Standard Methods for the Examination of Water and Wastewater (1998)


THAMNOTOXKIT (24 h, Acute Thamnocephalus platyurus toxicity test, fresh water) 

ARTOXKIT M (24 h, Acute Artemia franciscana toxicity test, estuarine/marine water)

direct toxicity artemia toxicitytestingThese two tests measure the acute mortality of Thamnocephalus platyurus and Artemia franciscana, two important small crustaceans, after a 24 h exposure. The test method involves hatching the organisms from dormant eggs, exposing them to a range of sample concentrations, and counting the number of dead after 24 hours. The THAMNOTOX test utilizes fresh water samples and the ARTOXKIT test utilizes estuarine or marine water samples. The assay is used to calculate the 24 h LC50 value.

  • The tests are highly reproducible and ensure uniform exposure conditions
  • The tests are cost and time effective
  • The organisms demonstrate increased sensitivity compared to other crustaceans
  • Have been used extensively in research for many publications and regulatory environmental testing


RAPIDTOXKIT (60 min, Sub-lethal Thamnocephalus platyurus toxicity test, fresh water)

This rapid test measures the ability of the crustacean Thamnocephalus platyurus to ingest red microbeads, similarly to the Rotofer assays. Exposure of the organisms to various test solutions at different concentrations is followed by application of the microbeads to the test solutions. At the termination of the assay, the ability of the Thamnocephalus to ingest the microbeads is measured by microscopy and the amount of organisms with red digestive tracts who have ingested the beads is the assay endpoint. The inability to perform this task is considered a measure of toxicity.

  • Assay is easy and incredibly fast to run, results are obtained the same day
  • Sensitivity is very high and the end point is sub-lethal.
  • More sensitive than bacterial luminescence inhibition tests for particular chemical compounds and biotoxins.
  • Detection threshold for toxicants is below Human Lethal Dose (HLD)
  • Particularly suited for assessment of (accidental or deliberate) water contamination threats.


TK33 - DAPHTOXKIT F magna (48 h, Acute Daphnia magna toxicity test, fresh water)

direct toxicity daphnia magna testThis test measures the acute mortality of Daphnia magna, an important, small freshwater crustaceans after a 48 h exposure. The test method involves hatching the organisms from dormant eggs, exposing them to a range of concentrations and counting the number of dead or immobile organisms after 48 hours.

  • Highly reproducible, cost- and time-effective
  • Comparable sensitivity to Daphnids from laboratory cultures
  • Daphnia are used worldwide for environmental screening
  • Acute Daphnia tests adhere to standard protocols developed for international organizations like the OECD and the ISO


CERIODAPHTOXKIT F (24 h, Acute Ceriodaphnia dubia toxicity test, fresh water)

Like the Daphnia magna test, Ceriodaphnia toxicity is an acute toxicity test with the freshwater crustacean Ceriodaphnia dubia. This assay also involves the growth and hatching of the organisms followed by exposure to different concentrations of the test compound dissolved in a water sample. Mortality is counted after 24 h as the assay endpoint, and the 24 h LC50 is calculated.

  • This assay is cost and time effective
  • The endpoints are easy to calculate
  • The assay produces repeatable results


OSTRACODTOXKIT F (6 day, Chronic Heterocypris incongruens toxicity test, fresh water)

direct toxicity ostracod testThis chronic toxicity test is unique, as it allows measurement of toxicity for fresh water sediment. The organisms are hatched, grown, fed, and exposed to a microenvironment containing the test sediment and an algae food source. Two endpoints are measured at the end of the 6 day incubation: mortality as well as turbidity of the water source (optical density, OD) as this represents the activity of the organisms as they ingest their food source.

  • This assay is highly specialized for sediment testing
  • The results reproduce well, and the assay is easy to score which reduces errors
  • The sensitivity compares favorably with that of “conventional” sediment contact tests e.g. amplipod Hyalella azteca.


ALGALTOXKIT F (72 h, Chronic Selenastrum capricornutum toxicity test, fresh water)

ALGALTOXKIT M (72 h, Chronic Phaeodactylum tricornutum toxicity test, estuarine/marine water)

direct toxicity test algae testBoth of these assays involve the growth of an algae culture and the exposure of the organisms to various concentrations of a test compound. Optical density is again used as the endpoint for these assays and the growth maintenance, as well as the overall health of the test populations is measured by the relative turbidity of the sample container. The assay can be performed in fresh or marine water and the results give essential toxicity data for a keystone species in many aquatic food chains.

  • The assays are reproducible and time effective
  • Demonstrate comparable sensitivity to the algal growth inhibition test with micro-algae from laboratory cultures
  • Used in many laboratories worldwide for routine screening of chemicals and environmental samples
  • A substantial number of publications have used these assays to report their data
  • The ALGALTOXKIT assays strictly adhere to protocols for regulatory testing with micro-algae, as described by international organizations e.g. OECD and ISO.


PHYTOTOXKIT (3-5 day, Chronic growth effect test on Sorghum saccharatum, Lepidium sativum and Sinapis alba, in sediment and soil samples)

direct toxicity plant testThis test measures the toxicity of contaminated soils, sludges, sediments, and pure compounds by assessing the germination ability and root growth of three higher order plants (1 monocotyl and 2 dicotyls) while exposed to the material in question. The end point is a visual inspection of the physical traits of the plants grown in the soil and is taken after a minimum of three days germination time.

  • The measurement endpoints are easy to assess and multiple tests can be carried out simultaneously.
  • The methodology is highly standardized and conforms to ISO 11269-1 procedure


PHYTOTESTKIT (3-5 day, Direct exposure, Chronic growth effect toxicity test on Sorghum saccharatum, Lepidium sativum and Sinapis alba)

This test is similar to the PHYTOTOXKIT test, which determines the effects of growth inhibiting chemicals present in soils and sediments and growth promoting chemicals on the germination and early growth of plants. However the PYTOTESTKIT assay does not use a soil vector and therefore measures the direct (intrinsic) effects of the compound under study on the plant populations.

  • This assay measures direct effects
  • The assay produces easily measurable endpoints and multiple compounds can be tested simultaneously
  • Exposure concentration can be more easily controlled.


SPIRODELA DUCKWEED TOXKIT (72 h, Direct Exposure, chromic growth effect toxicity test on Spirodela polyrhiza)

Presently there is only one toxicity test with a “floating” aquatic plant to assess the hazard of toxicants to higher plants, i.e. the assay with duckweeds Lemna minor or Lemna gibba. Standard test procedures for these duckweed assays have been published by national and international organizations, and Lemna tests are regularly used in the European Union for the authorization of Plant Protection Products (herbicides and plant regulators). This test procedure consists of 3 days germination of the organisms in a Petri dish, followed by exposure to different toxicant concentrations. The areas of the first fronds are measured at the start and at the end of the assay by image analysis on the 2 photos of the multiwells taken with a digital camera and transferred to a computer file. The growth inhibition of the first fronds is then calculated as the difference in the mean size of the first fronds at the start and at the end of the test.

  • Assay is unique for floating plants and requited for pesticide regulation
  • Is sensitive, with comparable sensitivities to older, more expensive Lemna assays
  • Is reliable and robust, has been tested extensively in multiple countries.