Biological test method for determining acute lethality of sediment to amphipods: chapter 2

Section 2: Test Organisms

• 2.1 Choosing Species
• 2.2 Life Stage, Size, and Source
• 2.3 Collection, Handling, and Transport
• 2.4 Holding and Acclimation
• 2.5 Selection of Test Organisms
• 2.6 Species-specific Application Limits

2.1 Choosing Species

One or more of the following species of marine or estuarine infaunal amphipods must be used with this reference method:

• Rhepoxynius abronius,
• Eohaustorius washingtonianus,
• Eohaustorius estuarius, or
• Amphiporeia virginiana.

Selection of one or more of these four species for use in a particular study must take into consideration the known or anticipated physicochemical characteristics of the test material (e.g., sediment grain size, porewater salinity, and porewater ammonia concentration) together with the known tolerance limits of the four candidate species to these characteristics. An investigator must be familiar with and/or consult the species-specific guidance provided in Appendices D (for R. abronius), E (for E. washingtonianus), F (for E. estuarius) and G (for A. virginiana) on the known tolerance and application limits for these four species, when choosing one to use in a test. In particular, the investigator should be aware that certain characteristics of each sample of test material to be evaluated using this reference method (namely, grain size and porewater salinity) must be within the species-specific application limits for these variables (see Section 2.6). Accordingly, the grain-size characteristics and porewater salinity of the test material needs to be known before choosing the test species. The investigator should also be aware of the known tolerance limits of each of these species to porewater ammonia (see Appendices D to G), and take this information into account together with that regarding known or anticipated concentrations of this contaminant in the test sediments, when choosing the test species and interpreting the findings of the test. Further guidance on the selection of test species is found in Section 2.1 of Environment Canada (1992).

2.2 Life Stage, Size, and Source

Juvenile or adult animals representing each of these species, which measure 3 to 5 mm total length, are available year round (EC, 1992) and should be used for this test. Very large individuals (i.e., those >5 mm total length) must not be used. Organisms that are too small to be retained on a 0.5-mm mesh screen should not be used.

All amphipods used in a test must be derived from the same population and source. In Environment Canada (1992), Appendices E (for A. virginiana), G (for E. estuarius), H (for E. washingtonianus), and K (for R. abronius) provide guidance on the appearance, behaviour, and distribution (including possible collection sites) of the species of amphipods to be used with this reference method. Sources commonly used for collection of test organisms include: West Beach, Whidbey Island, WA for R. abronius; Witty’s Lagoon or the exposed side of Esquimalt Lagoon, Victoria, BC for E. washingtonianus; Beaver Creek, Newport, OR for E. estuarius; and Martinique Beach, Halifax County, NS for A. virginiana. There are commercial vendors experienced in collecting and shipping these species. Laboratory personnel should be confident that any person(s) undertaking the collection, handling, and transport of organisms to be used with this reference method is/are fully familiar with and follow(s) recognized practices in these respects (EC, 1992; USEPA, 1994a), and that the organisms provided are indeed the correct species. For further information, contact Environment Canada or other members of the Scientific Advisory Group (Appendix C).

2.3 Collection, Handling, and Transport

Guidance given in Section 2.4 of Environment Canada (1992) should be followed when collecting, handling, and transporting amphipods. It is critical that standard, proven procedures be used to ensure that consistently healthy animals are obtained for the toxicity tests.

Containers used to transport amphipods are usually those used to hold and acclimate the organisms at the laboratory. Suitable containers with sealable lids include plastic food containers or plastic pails. At the collection site, a minimum 2-to 4-cm (or thicker) layer of sieved (0.5-to 1.0-mm mesh screen) sediment from the place where the animals are collected should be placed in the bottom of the container. Water from the collection site is then added to form a layer of ≥2 cm of overlying water. Amphipods sieved from other aliquots of the collection site sediment should then be transferred gently to the container. The density of amphipods in the container should not exceed 1 amphipod/cm² (USEPA, 1994a).

An appropriate quantity of sediment should be collected, sieved, and transported with the animals, for use as control sediment in the sediment toxicity test.

Long-distance transport (i.e., by air shipment) of E. washingtonianus or A. virginiana is not recommended because of unacceptably high mortality during holding and acclimation, or in control sediment during the test. An investigator may choose to do so rather than using an alternate species (i.e., R. abronius or E. estuarius); however, the species-specific criterion for a valid test (Section 4.6) defined herein must be met if the results of the test are to be considered valid and acceptable according to this reference method.

2.4 Holding and Acclimation

Guidance provided in Section 2.5 of Environment Canada (1992) should be followed when holding and acclimating each group of amphipods that has been collected for use in a sediment toxicity test.

Field-collected animals must be acclimated to the lighting, temperature, and salinity (for water overlying sediment) conditions to be applied during the test (see Section 4), for a minimum of two days and a maximum of ten days before the start of the test. Additionally, animals should not be held at the test facility for more than ten days following their collection, before the test is initiated.

Upon receipt of field-collected animals at the laboratory, the quality (i.e., temperature, salinity, dissolved oxygen, and pH) of the overlying water in one or more of the containers holding field-collected animals and sediment from the collection site should be determined and recorded. Any dead organisms observed on the surface of the sediment should be counted and removed, together with any debris evident. Sieving of the sediment in the container at any time before the day that the test is started is not recommended, since this procedure could unduly stress the test organisms. To minimize disturbance, amphipods should be held and acclimated in the container(s) used to collect and transport them. Alternatively, the organisms and sediment within the collection container(s) could be transferred (without sieving) to a larger holding/acclimation chamber if considered necessary to reduce crowding and increase surface area.

During the holding and acclimation period, amphipods should be held unfed in a minimum 2-to 4-cm (or deeper) layer of sediment from the collection site. Water overlying this sediment should be at least 2 cm deep. The dissolved oxygen content of this water must be maintained at 90 to 100% saturation by aeration or, if applied, by continuous replacement with oxygen-saturated water. Depending on the duration of the holding/acclimation period, the overlying water should be replaced continuously or periodically (e.g., daily) with air-saturated, fresh seawater adjusted to the required temperature and salinity.

During the holding and acclimation period, lighting must be constant and continuous. Overhead broad-spectrum (fluorescent or equivalent) lights should be used. Light intensity adjacent to the surface of the overlying water in the holding/acclimation container(s) should be 500 to 1000 lux.

The temperature of the overlying water must be adjusted gradually (i.e., no more than 3°C change per day) to a daily-mean acclimation temperature of 15 ± 2°C if acclimating R. abronius, E. washingtonianus, or E. estuarius; and to 10 ± 2°C if acclimating A. virginiana. Thereafter, amphipods must be held at the same respective temperature for a minimum of two days before their use in a test.

The salinity of the overlying water must be adjusted gradually (i.e., no more than 5‰ change per day) to a value representative of that of the porewater salinity measured for the sample(s) of test material. Thereafter, amphipods must be held at this salinity for a minimum of two days before their use in a test. In instances where a number of samples of test material (e.g., from different sampling stations and/or depths) are to be tested concurrently, the salinity of the overlying water must be adjusted to the mean porewater salinity determined for these samples (Section 4.3). The salinity to which test organisms are acclimated must be within their (species-specific) application limits (see Section 2.6). To minimize the interval between collection of test organisms and the start of the test, the necessary salinity and temperature adjustments (to acclimation and test conditions) may be conducted concurrently.

The temperature and salinity of the overlying water in each holding/acclimation chamber should be measured at least daily during any initial period of adjustment. Thereafter, the temperature, salinity, pH, and dissolved oxygen concentration in the overlying water must be measured at the beginning and end of the remaining period of acclimation (i.e., 2 to 10 days), as a minimum. It is recommended that temperature and salinity be measured daily during this period.

Water used to hold and acclimate test organisms may be that from an uncontaminated supply of natural seawater or reconstituted seawater. Guidance provided in Section 2.5.4 of Environment Canada (1992) should be consulted and followed when preparing and storing this water, and for monitoring its quality.

2.5 Selection of Test Organisms

The appearance and behaviour of amphipods in each holding/acclimation container should be “normal” and typical of the species (see EC, 1992). Any animal that fails to burrow in the holding sediment, or that appears or behaves atypically during the holding/acclimation period, must be discarded. Additionally, any animals that appear or behave atypically (see Section 2.2) when they are sieved from the collection-site sediment on the day that the test is started (Section 4.7) must be discarded. Records should be kept of the number of amphipods seen on the surface of the sediment or in the overlying water during the holding and acclimation period. The number of dead or atypical animals removed from each holding/acclimation container should also be recorded, for each period of observation.

2.6 Species-specific Application Limits

The physicochemical characteristics of each test sediment must be known before the species of test organism is selected. The choice of amphipod species to be used in a particular sediment toxicity test depends on the porewater salinity and grain size characteristics of the test material. The species-specific application limits in Table 1 must be followed, when choosing the test species as well as when acclimating them and undertaking the sediment toxicity test according to this reference method (see Appendices D, E, F, and G for details).

Table 1 Species-specific Application Limits for Reference Method