Zee- en zoetwater bioassays

The Algal growth inhibition test is performed in accordance with ISO guideline 10253 (ISO, 2006). A fixed number of algal cells from an exponentially growing mono-culture, are exposed to different concentrations of a test substance. All concentrations are tested in triplet. Nutrient medium is added to facilitate unrestricted growth in the test systems. Growth of the algae is assessed after 24, 48 and 72 h, using fluorescence measurements. The test is executed in a temperature controlled shaking cabinet with a continuous illumination.

The growth rate (r) is calculated for each replicate separately, by fitting the growth curves. Growth inhibition is calculated as percentage of the growth rate in the controls.

The inhibition percentages are plotted against the logarithm of the test concentrations. A non-linear model with variable slope is used to calculate the EC50 and EC10.

The test can be used to assess surface waters, effluents, sediment elutriates, sediment pore waters and extracts, and specific chemicals.

Different algal species are available for marine, as well as freshwater testing.

When only small amounts of sample are available, an algal growth inhibition test can be executed in a 96-well plate. For each sample less than 10 mL is needed if a concentration series is tested. Limit tests even acquire less sample volume.

Algae from an exponentially growing culture are introduced into the test vessels in a known density. The test vessels are incubated in a temperature controlled shaking cabinet at a fixed temperature with continuous illumination. Each 24h, the density is measured. After 72h, the test is terminated. Growth is expressed as specific growth rate (r). The effect is expressed as inhibition relative to control growth.

The density of the algae is determined using fluorescence measurements and is validated by cell counts.

The test can be used to assess surface waters, effluents, sediment elutriates, sediment pore waters and extracts, and specific chemicals. It is especially suited for test of for instance XAD-extracts.

Different algal species are available for marine, as well as freshwater testing.

In the Microtox® Basic test, luminescent bacteria Vibrio fisheri is exposed to solutions of test substances or effluents. When the bacteria are affected, the luminescence decreases in a dose-dependent manner.

Lyophilized bacteria are reconstituted in a reconstitution solution and transferred to the test vessels for acclimatization. The luminescence of the bacteria is then measured followed by adding test solutions in a concentration series. After fixed incubation times (usually 5, 15 and/or 30 min.), luminescence is registered. The changes in luminescence measured in the concentration series are compared to a control and when a dose-response curve is present an EC50 can be calculated.

The Microtox® Basic test is a rapid screening method for aquatic toxicity, suitable for surface waters, effluents, pore water and sediment extracts.

Rotifers are small planktonic animals, feeding on microalgae (phytoplankton). They are a major part of the zooplankton. When the environmental conditions become harsh (eg. in winter) they form cysts that can survive for prolonged periods as a resting stage. When the conditions improve, the cysts hatch and larvae develop into new rotifers. Cysts can be stored dry for long time. This feature has been used to develop standard test kits using dried rotifer cysts that can be hatched to provide synchronized larvae for testing purposes.

For tests with freshwater samples, we use the Rotoxkit F, using Brachyonus calyciflorus
For test with marine samples, we use the Rotoxkit M, using Brachyonus plicatilis

The test organisms, newly hatched nauplii larvae, are exposed to a concentration series of the test substance in 4 replicates of 5 animals. After 24h exposure, survival is assessed with a dissection microscope. An organism not showing any motility during 10 sec. observation is considered to be dead. The test is performed in darkness, in a temperature controlled cabinet at 25°C.

Mortality is plotted against the logarithm of the test concentrations. A non-linear model with variable slope is used to calculate the EC50 and EC10.

Dried cysts of the brine shrimp Artemia franciscana (formerly known as A. salina) can be stored for long times. By soaking the cysts in sea water, hatching occurs within 24 hours, resulting in a synchronised batch of nauplius larvae suitable for testing purposes.

For the tests, 2nd and 3rd instar larvae are exposed to different concentrations of a test substance. Each concentration is tested in triplet, using 10 organisms per test well. After 24 h exposure, survival of the test organisms is assessed with a dissection microscope. An organism not showing any motility during 10 sec. observation is considered to be dead. The test is performed in darkness, in a temperature controlled cabinet at 25°C.

Certified batches of cysts are used for the tests.

Mortality is plotted against the logarithm of the test concentrations. A non-linear model with variable slope is used to calculate the EC50 and EC10.

Daphnia magna acute and chronic test

The principle of this test is based upon USEPA guideline EPA/600/R-95-136 and RIKZ guideline SPECIE-05.

Conditioned oysters are obtained from a commercial nursery. The gametes are stripped and eggs are fertilized under controlled conditions. The resulting embryos are exposed to test concentrations for 48 hours at 20°C without light in a temperature controlled cabinet.

After 48 hours, a sub-sample of the test vessels is taken and the numbers of developed embryos are counted. Normally, straight-hinge D-larvae have developed within the time span. Development in test concentrations is expressed relative to control development.

Development is plotted against the logarithm of the test concentrations. A non-linear model with variable slope is used to calculate the EC50 and EC10.

The test is suitable for aquatic exposure within a salinity range of 20-35 ‰

The principle of this test is based upon OECD guideline 203 (1992). Common sole (Solea solea) is chosen as test organism, as it is an economically very important species in the North Sea region.

Freshly hatched yolk-sac larvae are exposed for 96 hours to concentrations of a test substance. All concentrations are tested in triplet, using 10 organisms per test vessel. The test vessels are incubated in a temperature controlled cabinet at 12°C, with a 16h/8h day/night regime for illumination. After 96 hours, the number of surviving larvae is counted under a dissection microscope. The surviving larvae are then narcotized and killed by freezing at -20°C.

Mortality is plotted against the logarithm of the test concentrations. A non-linear model with variable slope is used to calculate the EC50 and EC10.

In the Microtox® Basic test, the luminescent bacteria Vibrio fisheri is exposed to sediments. When the bacteria are affected, the luminescence decreases in a dose-dependent manner.

Lyophilized bacteria are reconstituted with a reconstitution solution. After acclimatization, the bacteria are mixed with different amounts of the test sediment and 3,5% NaCl-solution. After a fixed period, the sediment is removed by filtration and the water fraction is placed in an incubation block for stabilization. Then, the luminescence of the bacteria is measured and compared to a control without sediment. The change in luminescence is registered and a dose-response curve is obtained from which an EC50 can be calculated.

The Microtox® Solid Phase test is a direct contact test, suitable for coarse grained sediment, with a low silt fraction.

The test procedure is based upon RIKZ guideline SPECIE-02.

Amphipod 10d survival test

Amphipods are widely used for ecotoxicological testing of sediments. The mudshrimp Corophium volutator is a widely distributed estuarine species in North-western Europe. It is recommended as test species for sediment reworker test by OSPAR.

Mudshrimps are collected in the Wadden Sea, by sieving the top layer of muddy sediments. After acclimatising, animals within a specified size range are exposed to test sediments in a climate controlled room at 15°C with continuous illumination. The test sediments are 500 µm sieved prior to testing, in order to remove indigenous animals (predators and ‘wild’ mudshrimps).

After 10 days exposure, the test sediments are sieved again and the surviving mudshrimps are collected and counted.

The effect of test sediment is expressed as percentage mortality relative to the control (sediment of the collection area).

The test protocol is based upon RIKZ guideline SPECIE-01.

Related info: P5, P6

The lugworm Arenicola marina is a conveyor belt-type, sediment-feeding polychaete marine worm. It is found in high densities on sandy tidal flats. It feeds by ingesting the sediment at a depth of approx. 20 cm. Digested sediment is deposited on the sediment surface in characteristic casts. Daily cast production is used as a measure of feeding activity in this test.

Lugworms are introduced to test systems with a 3 cm thick layer of test sediment, covered with a thin layer of water. The water is continuously aerated. Produced casts are counted daily, after which the sediment surface is smoothed again.

The effect of the test sediments is expressed as percentage relative to cast production in a control sediment.

This species may also be used to assess the bioavailability of contaminants in sandy sediments, using a 28 d bioaccumulation test (see B12), or exposure in mesocosms (P5).

The ragworm Nereis virens is a predatory polychaete worm. It lives in a burrow in the sediment from where it undertakes its feeding activities. It feeds on small benthic animals and carrion.
It’s benthic mode of life makes it a suitable organism to study bioavailability of sediment-borne contaminants.

Ragworms are obtained from a commercial bait producer. The animals are exposed to test sediments in test vessels with 400 mL of the test sediment and 500 mL of sea water for 28 days in a climate room at 15°C with continuous illumination. The test vessels are gently aerated. The animals are fed once a week.
After an exposure period of 28 days, the animals are retrieved and stored in clean sea water overnight to purge their guts. The animals are then frozen at -20°C for chemical analyses.

When small animals (<3 gr) are used, also the effect of the test sediment on growth can be assessed. Small animals are, however, only available during a confined period in spring. Also the amount of material resulting may not be sufficient for some chemical analyses.

This test is especially applicable for very silty sediments. In sandy sediments, the lugworm Arenicola marina, may be more relevant since it is a true sediment-feeder