Toxicity of sewage effluent to marine invertebrates, tested at Davis 2009/10

Brief description

Two three-week toxicity tests were completed at Davis station 2009/10 as part of STP project 3217, to provide environmental information in support of an operational infrastructure project to up-grade sewage treatment at Davis (Project 3157). These tests addressed the third specific objective of the STP projects proposal; to determine the toxicity of sewage effluent as the basis for recommendations on the required level of treatment and on what constitutes an adequate or 'safe' dilution factor for dispersal of the effluent discharge to the near shore marine environment. Results from toxicity tests will provide a baseline by which future changes and/or improvements to the effluent discharge can be quantified (in terms of reduced toxicity and impacts to marine biota), if secondary treatment is re-established.

The toxicity of the sewage effluent discharge on marine invertebrates local to the Davis coastal area were assessed using standard bioassay protocols developed and tested under AAS #2933 (King). Two key local invertebrate species were used in both tests; the amphipod Paramoera walkeri and the microgastropod Skenella paludionoides. Invertebrates were exposed to varying concentrations of effluent collected from the outfall pipe (test 1) and from a composite made up of samples collected from the main holding tanks around Davis station (test 2). Each test included two controls with 0% effluent (seawater; SW and hyper-saline brine; HSB) and six concentrations of effluent; 3.125%, 6.25%, 12.5%, 25%, 50% and either 68% (test 1) or 63% (test 2). Dilution gradients of sewage effluent were used in tests in order to calculate the dilution of effluent required to cause no observable effects (NOEC), the lowest observable effect (LOEC) and predictable levels of response (e.g. ECX estimates) in test organisms. Organism responses are thereby used to derive protective concentration values for the effluent and the dilution required to have no predicted effects or impact on the community in the receiving marine environment. The behaviour and mortality of the test species were observed on day 1, 2, 4, 7, 10, 14 and 21 of the three week tests.

Test 1 (Discharged Effluent):
For test 1, effluent was collected directly from the station outfall line at 11am on 15/2/2010. Discharged effluent was collected in a 20 L bucket, placed at the end of the outfall line. Discharge of effluent from the outfall was initiated by pumping from the holding tank of the Sleeping and Medical Quarters / Old Living Quarters by the plumber (communication with the plumber via radio to co-ordinate the time of release from the tank). The effluent was decanted into containers on site and transported back to the laboratory for testing.

Test 2 (Composite Effluent):
For test 2, a 1 L sample of effluent was collected from each of the Davis station main building holding tanks, using a long handled ladle on 19/2/10. The composite provided an effluent sample that was representative of the waste generated in all activities on station from domestic activities and from work groups.

The volumes of each of the building effluents used to make the final composite test effluent were based on estimates of the volume of effluent created in each of the buildings as a proportion of the total stations effluent discharge and were as follows:

Building Name; as per map No. 14148,(Building Acronym),volume
Summer Accommodation Module,(SAM),416 mL
Temporary Accommodation Davis,(TAD),84 mL
Operations Building,(OPS),208 mL
Sleeping and Medical Quarters / Old Living Quarters,(SMQ/OLQ),834 mL
Living Quarters,(LQ),208 mL
Meteorology / Science Buildings,(MET/SCI),84 mL
Workshop,(WORKSHOP),84 mL
Climate Processes and Change,(ASP),84 mL

Location of buildings, within the Davis station area are provided in the Davis Buildings and Structures map (Map Catalogue No. 14148), available from the SCAR Map Catalogue at http://data.aad.gov.au/aadc/mapcat/display_map.cfm?map_id=14148.

Effluent dilution to test concentrations:
Effluent samples in both tests were adjusted to the required test salinity of 32.4 ppt using HSB to match the salinity of the ambient control SW collected off the coast off Davis and used as Control 1 in tests. The HSB salinity adjusted effluent was then diluted with SW to prepare the concentrations series of effluent (as effluent %) to be used as test solutions in toxicity tests. Each test included six concentrations of effluent; 3.125%, 6.25%, 12.5%, 25%, 50% and either 68% (test 1) or 63% (test 2).
Controls for both SW and HSB were also required as both were used as diluents in tests. The SW control used seawater from a field aquarium unit* (also the source of SW used to prepare effluent test solutions) and the HSB control was made using milliQ water that was adjusted to the test salinity using HSB. HSB was made by freezing and partially melting control seawater. The melt water is collected as hyper saline brine.

*The field aquarium unit contained locally sourced seawater, collected away from known contaminant sources. Water was physically filtered to 1 micron, biofiltered to remove ammonia, UV sterilised and held at a temperature of -0.8 degrees Celsius.

Physico-chemical analysis of test effulents:
Physico-chemical characteristics of each of the effluent samples including salinity, dissolved, oxygen, temperature and pH were measured immediately on return to the laboratory using a calibrated multi-meter.

Further characterisation of effluents, including coliform counts, microbial analysis, organic content and metal concentrations were conducted and are reported in other DAVIS_STP linked data sets.

Test species:
Two test species were used in both tests; the amphipod Paramoera walkeri and the microgastropod Skenella paludionoides. These test species were chosen based on their abundance and widespread distribution in nearshore environments around Davis Station.

Both species were collected by wading and dip netting from the shoreline at Airport Beach, an uncontaminated site located away from the current site of effluent discharge, and for microgastropods, additional sample were obtained from the surface of macro algae collected locally from boats. Both species were housed in control seawater in the field aquarium prior to their use in tests. Collection dates were 8/02/2010 for both species and for microgastropods also 2/02/2010 from Airport Beach (specific dates not known for other sites).

Toxicity Test Set up:
For each species, each test consisted of 4 replicate 70 mL vials per concentration (including 6 effluent concentrations and 2 controls) containing 60 mL of test solution. 10 individuals were added to each vial at the start of tests (total 320 individuals per species per test). An additional 10 individuals of each species was sampled and preserved in 100% ethanol for genetic analysis, and a further 10 individuals of each test species fixed in 4% formalin for taxonomy and size range analysis prior to the start of each test.

Test Conditions and maintenance:
Test vials were kept in culture cabinet at 0 degrees Celsius for the duration of the test. In tests with amphipods, a small strip of plastic mesh was added to each vial to provide a substrate and clinging surface for the amphipods. Invertebrates in test vials were fed and average of 0.036 g of Sera granumarin (granulated fish food) on day 6 of each week of the test. Test solutions were renewed in vials on day 7 of each week using freshly collected effluent for test 1 and a new freshly collected composite effluent for test 2.

Test Duration:
Observations of individuals in test were made at 24, 48, 96 hr, 7, 10, 14 and 21 days for each of the three week tests. This test duration was chosen based on its relevance to the rate of response of both species to effluent exposure. Individuals were scored as either alive or dead, and all dead invertebrates were removed immediately after scoring. A range of sublethal behavioural endpoints including activity were also investigated but did not reveal useful trends and were therefore not used in the final analyses. Test 1 commenced on 16/02/2010 and was terminated on 9/03/2010. Test 2 commenced on 19/02/2010 and was terminated on 12/03/2010.

Data Analysis:
For each species and each test, and at each of the 7 time end points (24, 48, 96 hr, 7, 10, 14 and 21 days), NOEC and LOEC values were determined using Dunnett's multiple comparison test. Probit Analysis or Trimmed Spearman Karber Tests with Abbott's correction (if assumptions of the Probit Analysis were not met) and Linear Interpolation with Bootstraping were also used to determine point estimates including EC1, 5, 10 and 50 values, and 95 % confidence limits (CL). If ECX, NOEC or LOEC values were outside the range of concentrations tested, results are reported as greater than (gt) or less than (lt) the highest or lowest concentration tested. All statistical analyses were done using the software Toxcalc for Excel (TidePool Scientific Software, California, 1992).

To determine the relative sensitivity of the 2 test species and the toxicity of the 2 effluent samples, EC50 values from tests were compared using ANOVA and SNK tests. An average EC50, NOEC and LOEC was determined for the effluent overall. To investigate the precision of the average EC50 estimate and the consistency of responses between tests, the coefficient of variation (CV) and 95 % CL of EC50 values among tests was also calculated.

Data Files Provided:
Three data files are provided with this record.
1. STP ECOTOX physico_chem data.xls
This file provides water quality measurements for effluent samples collected in association with the two toxicity tests (Test 1 and Test 2) for the three collection events (day 0, 7 and 14). Parameters measured include: pH, salinity, temperature, oxygen content, immediate oxygen demand (IOD) and FDO (FDO).
2. STPECOTOX Test 1 DISCHARGED.xlsx
This file provides toxicity test data (endpoint observations) for test 1, using effluent discharged from the Sleeping and Medical Quarters / Old Living Quarters outfall.
3. STPECOTOX Test 2 COMPOSITE.xlsx
This file provides toxicity test data (endpoint observations) for test 2, using a composite effluent collected from holding tanks of all Davis station building.