5    Sediment Toxicity


5.1    Background


In addition to the water quality monitoring programme, monitoring of sediment toxicity is recommended in the EIA to ensure that the disposal activities are not causing sediments adjacent to the pits of the mud disposal facility to become toxic to marine life.  This programme will employ standard techniques for sediment toxicity testing which are detailed in the following sections of this Manual. 

The ecotoxicological testing programme shall feature a suite of tests that include three phylogenetically distinct species that interact with bedded sediments in different ways.  Unacceptable impacts will have occurred if the levels of contaminants in the sediments collected in the area of the active pits are shown to have caused toxicity to marine fauna.  The findings of the sediment toxicity tests shall be compared to the results of the sediments chemistry.


5.2    Objective


The objective of this task is to determine if there are any changes and/or trends caused by disposal activities in the toxicity of sediments adjacent to the pits as a result of disposal activities.


5.3    Hypothesis


In accordance with the prediction of the EIA and the objectives of the Study, the impact hypothesis for this task shall be as follows:

There is no increase in sediment toxicity over time at individual stations or a trend of increasing toxicity with proximity to the pit.

The null hypothesis which should be statistically tested is as follows:


H0      Sediment collected at sites adjacent to the active pits exhibits no greater toxicity than sediment collected at sites remote from the facility. 


5.4    Sampling Design


In order to determine whether contaminated sediment placed in the active pits represents an ecological risk to biota in areas adjacent to the mud pit, ecotoxicological evaluations shall be performed on sediment collected from these surrounding areas.   

The toxicological testing programme should feature a suite of tests that includes three phylogenetically distinct species that interact with bedded sediments in different ways.  The testing programme shall include whole-sediment, or solid-phase toxicity tests using the epibenthic amphipod, Ampelisca abdita, the burrowing polychaete, Neanthes arenaceodentata, and free-swimming larvae of bivalves (Mytilus galloprovincialis) or other equivalent species.  The species to be used, either international or local, for the toxicity testing should be confirmed with EPD/AFCD before commencement of the testing.   

The experimental designs for assessing the impacts of disposal of contaminated mud in either the East of Sha Chau facility or the South Brothers facility on the toxicity of sediments in remote and adjacent areas take into account the following factors:


·                The null hypotheses being tested;

·                Location of other potential sources of contaminants in the North Lantau region, eg, Pearl River;

·                Predictions taken from the EIA on sediment plume locations; and,

·                Expected statistical treatment of the data.


Sediment samples shall be collected from two treatment areas.  The first treatment is represented by samples taken from two stations in an area close to the active pits (Near-Field) and the second treatment is represented by samples collected from stations in a reference area (Far-Field) (Figure 5.4a and 5.4b).  At each of the stations, five replicate composite grab samples shall be taken and used for the sediment toxicity tests.  The stations shall be sampled twice per year (wet season and dry season).  The sediment sampling shall be carried out when the pits are active.  The precise positions of the sampling stations should be confirmed in advance of the commencement of disposal activities and agreed with EPD based on the detailed design of the disposal facility.


5.5    Statistical Treatment of Data


Each of the toxicological tests shall be evaluated for statistically significant increases in toxicity.  Statistically significant toxicity shall be determined by performing an analysis of variance (ANOVA) test that compares the responses observed in the test treatments with those of the reference treatments.  At the end of the monitoring programme changes in the toxicity of the sediments over time shall be evaluated through the use of two-factor ANOVA incorporating both spatial and temporal scales of variation.

5.6    Use of Data


Once the data have been evaluated for significance, it is important to identify potential causes of toxicity and the biological significance of the observed effects.  The cause of the observed effects needs to be distinguished between 1) non-persistent contaminants, 2) persistent contaminants, and 3) physical factors.  It is most important to determine if the cause of the toxicity is due to persistent contaminants that are derived from the contaminated sediment placed in the pits (eg metals, pesticides, PAHs, TBT), to non-persistent contaminants (eg sulfides, ammonia, salinity) or to physical factors (eg grain size).

If the toxicity is due to persistent contaminants that are associated with disposal operations, the operations plan for the active pitsmay not be effective enough at managing the containment of contaminated sediment to acceptable levels and thus should be modified.  If the observed toxicity is due to non-persistent contaminants, the effects may be due to the pit but they are transient.  The toxicity of these types of contaminants can be assimilated by the environment in relatively short time periods, and are thus less harmful.  If the effects are related to physical factors, they are again of less concern and would not likely require changes in the facility operations plan. 


As non-contaminant factors and physical factors can confound toxicity test interpretation, the ET shall monitor ammonia, sulfides, interstitial salinity, and sediment-grain size.  Each of these factors has been observed to elicit a Atoxic@ response in test organisms, however, they are not factors related to persistent contaminants of concern.  This information shall be used to investigate any observed toxicity responses and determine whether the response is due to persistent contaminants or to more transient factors.


5.7    Data Collection Parameters


The amphipod toxicity test with Ampelisca abdita (or a equivalent species as agreed with EPD/AFCD prior to conduct of the toxicity test) shall evaluate survival following a 10-day exposure to test sediment.  Procedures shall follow those outlined in PSEP (1995) ([1]) and CEDD's Environmental Laboratory Guidance Document (1996) (2).  The amphipod benthic test shall be conducted as a static test and shall be performed with 175 ml of sediment and 800 ml of overlying seawater placed in a 1-L glass jar.  At test initiation, each of five replicate test chambers shall be seeded with 20 amphipods.  Test chambers shall be maintained at 20°C and shall be checked daily throughout the test to establish trends in sediment avoidance.  After the 10-day exposure, the benthic tests shall be terminated by sieving the sediments and enumerating the live and dead amphipods.


The test on Neanthes arenaceodentata (or a equivalent species as agreed with EPD/AFCD prior to conduct of the toxicity test) shall evaluate polychaete survival and growth following a 20-day exposure to test sediment.  Test methods will follow those outlined in PSEP (1995) (3).  The test shall be conducted as a static test, performed in 175 ml of sediment and 800 ml of overlying seawater in 1-L glass jars.  At test initiation, each of five replicate test chambers shall be seeded with five polychaetes.  Test chambers shall be maintained at 20°C and shall be checked daily to record mortality and sediment avoidance.  To promote growth, worms shall be fed TetraMarin8 every third day throughout the test.  After 20 days, the N. arenaceodentata test shall be terminated by sieving each test chamber and enumerating both live and dead organisms.  Surviving polychaetes shall be dried and weighed for each test chamber.  Average dry weight shall be compared to initial biomass to determine mean growth for each test chamber. 


The larval-development toxicity test shall be performed with fertilized bivalve embryos eg Mytilus galloprovincialis or a equivalent species as agreed with EPD/AFCD prior to conduct of the toxicity test, and shall evaluate larval survival and development following a 48 to 96-hour exposure to test sediments.  This procedure shall follow those outlined in PSEP (1995) ([1]).  This test shall be conducted in 20 mg of test sediment with 800 ml of seawater in 1-L glass jars.  At test initiation, test jars shall be seeded with 20 to 40 embryos per ml.  Test chambers shall be maintained at 16°C.  At termination, overlying water shall be decanted and subsamples drawn from the supernatant.  Survival and normal larval development shall then determined under an inverted compound microscope.


In each of the sediment tests, a native/seawater control (consisting of sediment from the amphipod or polychaete collection site or clean seawater for the larval test) shall be tested concurrently with the test sediments.  The control treatment should be included to determine the health of the test organisms.  Sediments collected from the reference stations shall also be tested concurrently with test sediments to provide a basis for statistical comparison.  For the larval tests, grain-size controls shall be tested concurrently with the test sediments to discern any effects related to sediment grain size.  Additionally, a water-only reference toxicant test using cadmium (from CdCl2) or copper (from CuNO3) shall be conducted with each batch of test organisms.  This reference-toxicant test provides a measure of relative sensitivity for each group of test organisms.  All toxicity tests shall be completed and reported within 2 months from collection of the samples.


Prior to the commencement of disposal activities, the data collection parameters and testing protocols described above shall be reviewed and revised where necessary and agreed with the relevant government departments (eg EPD/AFCD).


5.8    Sampling Procedure and Equipment


Procedures for sampling shall be as for the sediment chemistry for Sediment Quality Monitoring as detailed in Section 4.8 of this Manual.  Shipments of the sediments shall be packaged in ice-boxes in order to maintain the sediments at a constant temperature of 4oC and dispatched by express courier for immediate testing.


5.9    QA/QC


To ensure the quality and integrity of the ecotoxicological data and subsequent analyses, a QA/QC control program shall be followed that meets or exceeds the QA/QC program outlined in Chapter 4 of A Testing of Dredged Material for Marine Disposal:  Environment Laboratory Guidance Document.  The QA/QC program for the facility ecotoxicological program is described below.


5.9.1    Sediment Handling and Chain-Of-Custody


Upon sample receipt, samples shall be held at 4° ± 2° C in the dark until required for testing.  Sediment holding times for biological testing begin the day of sample collection and shall be kept at a minimum.  The holding time for sediment intended for biological testing shall be 6 weeks.  Chain-of-custody forms shall accompany each batch of samples to track samples and to provide temperature data before and after shipping. 


5.9.2    Bioassay Seawater


Clean seawater for holding test organisms shall be sand-filtered seawater piped directly into the testing laboratory.  Seawater used for test water and control water should be additionally gravity-feed filtered through a 0.45 -mm filter before use for all test species.  Bioassay seawater should be continually monitored for water quality and the presence of algal blooms.


5.9.3    Instrument Maintenance and Calibration


Procedures for calibration and maintenance of water quality equipment shall follow MSL protocols.  All measuring and testing equipment used on this Project should be traceable to the data collected and should be calibrated before use.

The pH meters used for obtaining water quality data must be calibrated daily before use according to MSL-M-045, Calibration and Use of pH Meters.  The calibration shall be documented on the pH Meter Calibration Record sheet.  Maintenance on pH meters shall be performed monthly.  Maintenance should include visual inspection, cleaning probes in 0.1 M HCl, and cleaning any corroded contacts.

Refractometers used for obtaining water quality data shall be calibrated monthly using IAPO Standard Seawater according to MSL-M-048, Calibration and Use of Refractometers.  The calibration should be documented on the Refractometer Calibration Record sheet.  Refractometers should be inspected visually and cleaned monthly.


Digital thermometer calibrations shall be performed monthly by comparison to a certified mercury thermometer as specified in MSL-M-047, Calibration and Use of Thermometers.  The calibration shall be documented on a Thermometer Calibration Record.  Maintenance should include visual inspection and cleaning of salt and corrosion from connectors and contacts.


Dissolved oxygen meters should be calibrated daily before use according to MSL-M-046, Calibration and Use of Dissolved Oxygen Meters.  The calibration should be documented on the Dissolved Oxygen Meter Calibration Record.  Maintenance should be performed once monthly and should include visual inspection, cleaning the probe, and replacing of probe membrane.


The Fisher Accumet 1003 pH/selective ion electrode meter with ammonia electrode should be maintained according to manufacturer’s instructions.  The meter should be calibrated on each day of use with three concentrations of NH4Cl standards bracketing the expected test concentrations of ammonia.  The ammonia probe should be stored in 0.02 M NH4Cl when not in use.


5.9.4    Data Review and Validation


In addition to QA/QC mentioned above, a series of reviews by qualified laboratory personnel should be implemented to ensure that the data generated for this Project meets the data quality objectives.  These reviews should include the following:


·                Data should be reviewed periodically by laboratory personnel to ensure that sample testing activities are completely and adequately documented. 

·                Sample holding times, sample integrity, test animal handling and acclimation, equipment calibration, water quality measurements, reference toxicity results, observations, and control survival shall be reviewed by qualified laboratory personnel.  The results of QC measurements shall be compared to pre-established criteria as a measure of data acceptability.

·                A final data audit by the Quality Assurance Officer shall be performed prior to submission of the data and report.  This audit will ensure that the data are accurate, traceable, defensible, and complete, as compared to the Manual.  The audit procedure (MSL-Q-005, Quality Assurance Data Audits) is a statistical, randomized check which involves comparing selected reported values to the original data.  This procedure is designed to ensure a 95 percent chance of detecting whether one percent or more reported values disagree with the original data.


The overall quality assurance objective for this Project is to implement procedures that will ensure the collection of representative data that is of acceptable and defensible quality.  The data quality objectives for the ecotoxicological tests shall be devised with reference to the previous data quality objectives established for the previous monitoring programmes for the East of Sha Chau CMPs.


A negative control provides a measure of test organism health.  Negative control treatment shall be running concurrent to each toxicity test as a measure of the test organism's health.  For the amphipod (eg Ampelisca sp ) and polychaete (eg Neanthes sp) toxicity tests, the negative control should consist of clean, native sediment that is to be collected from the test organism's natural habitat.  For the bivalve larval test, the negative control should consist of clean seawater.  Acceptable limits for the negative controls shall be defined with reference to the limits established for the East of Sha Chau CMP monitoring programmes.  If survival or normal development do not meet the acceptability criteria, all data should be evaluated and the test may need to be repeated.


Water quality measurements provide documentation of environmental conditions within the test chambers during the exposure.  Temperature, dissolved oxygen, pH, and salinity shall be measured daily throughout the test.  Conditions that are acceptable to maintain the health of the test organisms shall be defined with reference to the acceptable conditions defined for the East Sha Chau CMP monitoring programmes.  If test conditions are outside the acceptability criteria, the data will need to be qualified. 


The positive control provides a relative measure of test organism sensitivity.  For each of the bioassays for the active pits, a separate reference-toxicant test should be performed with each batch of test organisms.  The results of the reference-toxicant tests shall be compared with control charts generated by the testing laboratory for that species and toxicant.  Those results within two standard deviations of the cumulative mean are considered to be similar in sensitivity to previous test populations.  For amphipods (eg A. abdita) the reference-toxicant test shall be performed with cadmium in the form of cadmium chloride; for polychaetes (eg N. arenaceodentata) and bivalve larvae reference-toxicant tests shall be performed with copper as copper nitrate.  If the test results are outside the control limits, the data will need to be qualified.


([1])                         PSEP (Puget Sound Estuary Program) 1995  Recommended guidelines for conducting laboratory bioassays on Puget Sound sediments.  Prepared for the US EPA.

([3])  PSEP (1995) op cit.


([4])                         PSEP (1995) op cit