5.1 The ecological importance of bivalves, their wide geographic distribution, ease of handling in the laboratory and the field, and their ability to filter and ingest large volumes of water and sediment particles make them appropriate species for conducting field bioassays to assess bioaccumulation potential and associated biological effects. The test procedures in this guide are intended to provide guidance for conducting controlled experiments with caged bivalves under “natural,” site-specific conditions. It is important to acknowledge that a number of “natural” factors can affect bivalve growth and the accumulation of chemicals in their tissues (Section 6, Interferences). This field bioassay can also be conducted in conjunction with laboratory bioassays to help answer questions raised in the field exposures. The field exposures can also be used to validate the results of laboratory bioassays.

5.2 The ultimate resources of concern are communities. However, it is often difficult or impossible to adequately assess the ecological fitness or condition of the community or identify and test the most sensitive species. Bivalves are recommended as a surrogate test species for other species and communities for the following reasons: (1) They readily accumulate many chemicals and show sublethal effects associated with exposure to those chemicals (2); (2) they accumulate many chemicals through multiple pathways of exposure, including water, sediment, and food (24, 25, 26, 27, 28, 29), and (3) caged bivalves have been shown to represent effects on the benthos more accurately than traditional laboratory tests (30, 31). Although bivalve species might be considered insensitive because of their wide use as indicators of chemical bioavailability, it has been suggested that sensitivity is related to the type of test, end points being measured, and duration of exposure (2). In relatively short-term toxicity assessments in which survival is typically determined as the measurement end point, bivalves may appear to be more tolerant to and less affected by chemicals because of their ability to close their valves for short periods and avoid exposure (32, 33, 34, 35) . However, studies comparing the mortality end point in bivalves and other test species have found bivalves to be equally (36, 37) or more sensitive (38, 39) than the other species (Table 1). When the bivalve growth end point was compared to the mortality end point in other test species, the bivalve growth end point was more sensitive (20, 30, 31, 40, 41).

5.2.1 Chronic tests designed to monitor sublethal end points, such as growth, are recommended because bivalves generally show increasing sensitivity with increasing exposure period. Sublethal end points measured in bivalves that have demonstrated high levels of sensitivity include growth (3, 20), reproduction (21), DNA damage (42, 43), metallothioneins and other biochemical markers (44, 45, 46).

5.2.2 There are many field monitoring programs in the US which use bivalves, including the NOAA Status and Trends Program (47), the California Mussel Watch (48), and the California Toxics Monitoring Program, a freshwater monitoring program (49). Similar field-monitoring programs exist in other countries. Numerous laboratory studies throughout the world have examined bioaccumulation and biological effects in bivalves. The existing databases which have compiled bioaccumulation and effects in bivalves and other species (8, 9) make it possible to use tissue residues associated with effects in bivalves as surrogates to estimate effects in both water column and benthic organisms in many freshwater, estuarine, and marine environments.

5.3 Bivalves are an abundant component of many soft bottom marine, estuarine, and freshwater environments. Intertidal marine bivalves make up a significant portion of many habitats and provide habitats for many additional species. It is important to monitor freshwater bivalves for the following reasons: they are among the first taxa to disappear from benthic communities impacted by chemicals; they have been shown to be more sensitive than several other major taxa in laboratory tests.(50) The threatened and endangered status of many freshwater bivalve species also make them an important group to monitor.

5.4 If practical, the species to be used in a field bioassay should be one that is endemic to the area under investigation. In many cases, the specific area under investigation may not support bivalves due to a variety of factors including high concentrations of chemicals, competition or predation, or lack of suitable habitat or substrate. Under these conditions, it may be desirable to use a species that would normally be found in the environment if all conditions were favorable; however, it may be necessary to use a surrogate species, that is, a species that can tolerate the environmental conditions but is not normally found in the area, if native species are unavailable in the test area.

5.5 Bivalves generally utilize one of two primary modes of feeding: filter-feeding or deposit feeding. However, all known deposit-feeding bivalves are facultative in that they can either deposit- or filter-feed. Filter-feeders assimilate dissolved organics as well as suspended particulate matter, including plankton and suspended sediments, from the water column and have the potential for exposure to chemicals associated with this ingested material. Facultative deposit-feeding bivalves can be exposed to chemicals associated with sediments as they ingest sediments. They also ingest particulate material from the water column as they filter feed. As such, bivalves are capable of integrating exposure to chemicals dissolved in water and sorbed on sediment particles on the bottom or in suspension. It should be acknowledged that bivalves transplanted in the overlying water above sediment or transplanted directly on or in sediment may not exclusively accumulate or be affected by chemicals in a particular medium. That is, bivalves in or on sediment may still filter and accumulate chemicals from overlying water. Conversely, bivalves transplanted in the water column may filter suspended sediment and accumulate chemicals from that sediment. Bivalves can also assimilate chemicals as they ventilate overlying water.

5.6 Field bioassays are conducted to obtain information concerning the bioavailability of chemicals in the water column or bedded sediments and subsequent biological effects on bivalves after short- and long-term exposure to water and sediment under site-specific conditions. These bioassays do not necessarily provide information about whether delayed effects will occur, although a post-exposure observation period could provide such information. Sublethal post-exposure observations may include gonad development, spawning success, gamete survival, and development. The decision to conduct post-exposure studies in the field or in the laboratory depends on the observations being made, test conditions required, and experimental logistics.

5.7 The in-situ exposures described in this guide could be followed by laboratory measurements, such as scope for growth (2), filtration rate (51), byssal thread production (52, 53, 54), and biomarkers (55, 56).

5.8 The bivalve field bioassay can be used to determine the spatial or temporal trends of chemical bioavailability in water and sediment and effects due to exposure to those chemicals. Spatial comparisons of parameters of concern can be made by distributing the caged bivalves along physical and chemical gradients at scales commensurate with the desired level of discrimination. For example, station locations might be distributed along a known physical or chemical gradient in relation to the boundary of a disposal site (57, 58, 59, 60, 61), sewage outfall (62), or effluent pipe or at stations identified as containing elevated concentrations of chemicals in water or sediment as identified in a reconnaissance survey (3, 63, 64). This can be accomplished by placing caged bivalves along horizontal transects or at different depths in the water column. Temporal comparisons can be made by conducting before-and- after studies. For example, the effectiveness of dredge activities, effluent diffuser construction, effluent reduction, or remedial action can be determined by conducting field bioassays before the action, during the action, and after the action.

5.9 The relative bioavailability of chemicals from the various pathways of exposure (that is, aqueous phase, suspended particulate matter, sediment) and subsequent effects can be determined by simultaneously deploying bivalves with different feeding strategies and making supplementary measurements. A combination of filtration and the use of sediment traps followed by chemical analysis of the various environmental compartments can be used to identify the relative contribution of the aqueous phase, suspended particulate matter, and sediment. Lipid bags or semi-permeable membrane devices (SPMDs), which predominantly collect the dissolved fraction of chemicals, could also be used ( 65, 66, 67, 68, 69, 70). The bioaccumulation of chemicals and effects among different bivalve species deployed either side-by-side, at exposure and reference locations, or before and after exposures can be compared and used to help explain the spatial variability of chemical contamination, particularly if the different species are placed in different locations (that is, in the water column, on top of the sediments, within the sediments) as determined appropriate for the study design. This field assessment approach could be supplemented with laboratory studies designed to answer specific questions regarding dissolved versus particulate pathways of exposure.

5.10 Results of bivalve field bioassays might be an important consideration when assessing the hazards of materials to aquatic organisms (see Guide E1023) or when deriving water or sediment quality guidelines for aquatic organisms (17, 71). Bivalve field bioassays can be useful in making decisions regarding the extent of remedial action needed for contaminated sites. They also provide a convenient method for manipulative field experiments, hypothesis testing, and monitoring specific sites before, during, and after cleanup operations (63, 64).

1.1 This guide describes procedures for conducting controlled experiments with caged bivalves under field conditions. The purpose of this approach is to facilitate the simultaneous collection of field data to help characterize chemical exposure and associated biological effects in the same organism under environmentally realistic conditions. This approach of characterizing exposure and effects is consistent with the US EPA ecological risk assessment paradigm. Bivalves are useful test organisms for in-situ field bioassays because they (1) concentrate and integrate chemicals in their tissues and have a more limited ability to metabolize most chemicals than other species, (2) exhibit measurable sublethal effects associated with exposure to those chemicals, (3) provide paired tissue chemistry and response data which can be extrapolated to other species and trophic levels, (4) provide tissue chemistry data which can be used to estimate chemical exposure from water or sediment, and (5) facilitate controlled experimentation in the field with large sample sizes because they are easy to collect, cage, and measure (1, 2)2. The experimental control afforded by this approach can be used to place a large number of animals of a known size distribution in specific areas of concern to quantify exposure and effects over space and time within a clearly defined exposure period. Chemical exposure can be estimated by measuring the concentration of chemicals in water, sediment, or bivalve tissues, and effects can be estimated with survival, growth, and other sublethal end points (3). Although a number of assessments have been conducted using bivalves to characterize exposure by measuring tissue chemistry or associated biological effects, relatively few assessments have been conducted to characterize both exposure and biological effects simultaneously (2, 4, 5). This guide is specifically designed to help minimize the variability in tissue chemistry and response measurements by using a practical uniform size range and compartmentalized cages for multiple measurements on the same individuals.

1.2 The test is referred to as a field bioassay because it is conducted in the field and because it includes an element of relative chemical potency to satisfy the bioassay definition. Relative potency is established by comparing tissue concentrations with effects levels for various chemicals with toxicity and bioaccumulation end points (6, 7, 8, 9, 10) even though there may be more uncertainty associated with effects measurements in field studies. Various pathways of exposure can be evaluated because filter-feeding and deposit-feeding are the primary feeding strategies for bivalves. Filter-feeding bivalves may be best suited to evaluate the bioavailability and associated effects of chemicals in the water column (that is, dissolved and suspended particulates); deposit-feeding bivalves may be best suited to evaluate chemicals associated with sediments (11, 12, 13, 14). It may be difficult to demonstrate pathways of exposure under field conditions, particularly since filter-feeding bivalves can ingest suspended sediment and facultative deposit-feeding bivalves can switch between filter- and deposit feeding over relatively small temporal scales. Filter-feeding bivalves caged within 1 m of bottom sediment have also been used effectively in sediment assessments from depths of 10 to 650 m (5, 15, 16). Caged bivalve studies have also been conducted in the intertidal zone (17). The field testing procedures described here are useful for testing most bivalves although modifications may be necessary for a particular species.

1.3 These field testing procedures with caged bivalves are applicable to the environmental evaluation of water and sediment in marine, estuarine, and freshwater environments with almost any combination of chemicals, and methods are being developed to help interpret the environmental significance of accumulated chemicals (6, 7, 9, 18, 19). These procedures could be regarded as a guide to an exposure system to assess chemical bioavailability and toxicity under natural, site- specific conditions, where any clinical measurements are possible.

1.4 Tissue chemistry results from exposures can be reported in terms of concentrations of chemicals in bivalve tissues (for example, µg/g), amount (that is, weight or mass) of chemical per animal (for example, µg/animal), rate of uptake, or bioaccumulation factor (BAF, the ratio between the concentration of a chemical in bivalve tissues and the concentration in the external environment, including water, sediment, and food). Tissue chemistry results can only be used to calculate a BAF because caged bivalves in the field are exposed to multiple sources of chemicals and can accumulate chemicals from water, sediment, and food. Toxicity results can be reported in terms of survival (3, 20), growth rate (3, 20), or reproductive effects (21, 22) after a defined exposure period.

1.5 Other modifications of these procedures might be justified by special needs or circumstances. Although using appropriate procedures is more important than following prescribed procedures, results of tests conducted using unusual procedures are not likely to be comparable to results of standardized tests. Comparisons of results obtained using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting field bioassays with bivalves.

1.6 This guide is arranged as follows:

Section

Referenced Documents

 2

Terminology

 3

Summary of Guide

 4

Significance and Use

 5

Interferences

 6

Hazards

 7

Experimental Design

 8

Apparatus

 9

 Facilities

 Construction Materials

 Cages

Test Organisms

 10

 Species

 Commonly Used Taxa

 Size and Age of Test Organisms

 Source

 Number of Specimens

 Collection

 Handling

 Holding

 Animal Quality

Field Procedures

 11

 Test Initiation: Presort

 Final Measurements and Distribution

 Attachment of PVC Frames

 Deployment

 Retrieval and End-of-Test Measurements

 Analysis of Tissues for Background Contamination

 Decontamination

 Collection and Preparation of Tissues for Analysis

 Quality Assurance/Quality Control Procedures

 Sample Containers, Handling, and Preservation

Ancillary Methodology

 12

 Temperature

 Food

Acceptability of Test

 13

Report

 14

Keywords

 15

References

1.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.

1.8 This standard may involve hazardous materials, operations, and equipment – particularly during field operations in turbulent waters or extreme weather conditions. This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. Specific hazard statements are given in Section 7.

1.9 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.