Appendix A. Supplementary material

Assessing estuarine quality: A cost-effective in situ assay with amphipods

Monica Martinez-Haroab*, Pelayo Acevedob, Antónia Juliana Pais-Costaa, Mark A. Taggartc, Irene

Martinsa, Rui Ribeirod, João Carlos Marquesa

aMARE – Marine and Environmental Sciences Centre, Department of Life Sciences, University of

Coimbra, Portugal

bInstituto de Investigación en Recursos Cinegéticos, IREC (UCLM-CSIC-JCCM), Ronda de Toledo

12, Ciudad Real 13071, Spain

cEnvironmental Research Institute, University of the Highlands and Islands, Castle Street, Thurso,

Caithness, Scotland, KW14 7JD, UK

dCentre for Functional Ecology, Department of Life Sciences, University of Coimbra, Calçada

Martim de Freitas, 3000-456 Coimbra, Portugal

Corresponding author e-mail address: monica.martinezharo@gmail.com

Nº pages: 6

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UNIVERSITY OF COIMBRA MARINE AND ENVIRONMENTAL SCIENCES CENTRE (MARE-UC)

FACULTY OF SCIENCES AND TECHNOLOGY DEPARTMENT OF LIFE SCIENCES

STANDARD OPERATING PROCEDURE

Installation Date: May 2015 Version: 1.0 Status: Operational Number of pages: 6 Title: Assessing estuarine quality: A cost-effective in situ assay with amphipods

(i) PURPOSE: Standardization of the procedure for deployment and processing of in situ assays based on postexposure feeding of amphipods. (ii) SCOPE: To be applied in all in situ assays based on postexposure feeding utilizing amphipods Echinogammarus marinus in lower intertidal estuarine systems. (iii) REFERENCES: ASTM (American Society for Testing and Materials), 2002. Standard Guide for Conducting Acute

Toxicity on Test Materials with Fishes, Macroinvertebrates and amphibians. E 729-96. Philadelphia, PA, USA.

Guillard, R.R.L., 1983. Culture of phytoplankton for feeding marine invertebrates. In Berg CJ Jr, ed, Culture of Marine Invertebrates. Hutchinson-Ross, Stroudsberg, PA, USA, pp 108–132.

Martinez-Haro, M., Acevedo, P., Pais-Costa, A.J., Taggart, M.A., Martins, I., Ribeiro, R., Marques, J.C. 2016. Assessing estuarine quality: A cost-effective in situ assay with amphipods. Environmental Pollution DOI: 10.1016/j.envpol.2016.01.071.

Marques, J.C., Nogueira, A., 1991. Life cycle, population dynamics and production of Echinogammarus marinus (Leach) (Amphipoda) in the Mondego estuary (Portugal). Oceanol. Acta 11, 213–223.

(iv) PROCEDURE: A. MATERIALS 1.0 Materials for preparation of food for postexposure feeding quantification (per environmental site) 1 Artemia franciscana cysts vial 2 Plastic/glass diameter petri dish of approximately 10-cm diameter 1 Plastic/glass diameter petri dish of approximately 6-cm diameter 50 ml Reconstituted sea water (hereafter RSW) at 33-g/L salinity (Artificial Seawater Provasoli-McLachlan; ∼33 g/L salinity; J. McLachlan’s modification of the artificial seawater with L. Provasoli’s metal mix PI described in Guillard, 1983) 50 ml RSW at 20-g/L salinity 50 ml Reconstitute fresh water medium (ASTM, 2002) 1 Light source (approximately 100 µmole photons/m2/s) 1 Incubation cabinet/room at 25ºC 1 Pasteur pipette provided with a bulb

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1 Micropipette provided with a bulb 40 1.5-ml Plastic microtubes 1 Rack for 1.5-ml microtubes 1 Plastic box with cap/plastic bag 1 Stereomicroscope 1 Plastic bag 1 Freezer at -20ºC 2.0 Materials for selecting the adequate deployment day (per environmental site) 1 Tidal table, specific for the area under study 3.0 Materials for deployment (per environmental site) 3 Assay chambers (each composed by one transparent acrylic tube [20-cm long, 5-cm inner diameter, 6 cm outer diameter], and one cup with 200-µm nylon mesh; see Figure 1, Appendix) 4 100-ml Plastic containers 1 1-L RSW at 33-g/L salinity 40 Juveniles E. marinus (approximately 6-mm total body length) 1 3-ml Plastic pipettes with the tip cut 1 Thermally insulated box 1 Pair of rubber gloves 1 Pair of rubber fishing boots 4.0 Materials for the end of in situ assay (per environmental? site) 1 Pair of rubber gloves 1 Pair of rubber fishing boots 3 Corks (5-cm diameter) 3 1-L Plastic bags (approximately 15-cm length, 25-cm height) 2 5-L Plastic bottles 2 15-L Plastic bags 2 Thermally insulated box 5.0 Materials for postexposure feeding quantification (per environmental site) 35 1.5-ml Microtubes, each containing 60 Artemia nauplii 35 30-ml Glass beakers (approximately 6-cm height, 3-cm diameter) 500 ml RSW at 33-g/L salinity 3 Plastic/glass diameter petri dish of approximately 10-cm diameter 3 500-ml Plastic containers (approximately 10-cm height, 8-cm diameter) 4 White plastic trays (approximately 30-cm length, 20-cm height, 5-cm width) 1 1-L Wash-bottle 1 5-ml Micropipette 1 5-ml Micropipette tip 1 Pasteur pipette provided with a bulb 1 Micropipette provided with a bulb 1 3-ml Plastic pipettes with the tip cut 1 3-ml Plastic pipettes 1 Incubation room/cabinet at 20 ± 1°C in darkness 35 Plastic/glass petri dishes of approximately 6-cm diameter 1 Square of black cartoon (approximately 10-cm2) 1 Clock 1 Stereomicroscope 1 Battery of six sieves: 63, 125, 250, 500, 1000, and 2000 µm

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1 Sieve mechanical shaker (optional; e.g. Retsch VE 1000, Haan, Germany) 1 Oven at 50 ºC 1 Balance B. METHODS 1.0 Method for the preparation of food for postexposure feeding quantification (per environmental site) 1. Empty the contents of the vial with cysts into the 10-cm petri dish. 2. Fill the 10-cm petri dish with RSW at 33-g/L salinity up to approximately 0.5 cm from the top, swirl gently to distribute the cysts evenly and cover it. 3. Expose the petri dish to the light source and incubate it at 25ºC for 24 hours (approximate time needed for cyst hatching, though it can depends on the source of cysts). 4. Fill the other 10-cm petri dish with RSW at 20-g/L salinity. 5. Transfer newly hatched nauplii from the hatching petri dish to the petri dish filled with RSW at 20-g/L salinity using the Pasteur pipette with bulb. 6. Fill the 6-cm petri dish with reconstituted freshwater medium. 7. Transfer some newly hatched nauplii from the petri dish filled with RSW at 20-g/L to the 6-cm petri dish filled with freshwater medium using the Pasteur pipette. 8. Put the 6-cm petri dish under the stereomicroscope and transfer 60 nauplii into each of the 1.5-ml microtubes (placed on the respective rack for 1.5-ml microtubes) using the micropipette with bulb. 9. Store the rack with the 1.5-ml microtubes upright in the freezer at -20ºC until frozen. 10. Transfer the 1.5-ml to a plastic box with cap or a plastic bag with the respective tag until use. 2.0 Method for selecting the adequate deployment day (per environmental site) During the exposure period, fluctuations in water-column depth occur due to the effect of tides. The selection of the adequate deployment day is necessary to have a similar water depth on deployment and recovery days in order to find and retrieved the chambers successfully in the field after the exposure period. 1. Determine the adequate deployment day, using the tidal table, considering that: a) chambers must be exposed in the field during a 48-h period; b) during the exposure period, the low tide levels must be equal or higher than the low tide level registered at deployment; c) at retrieval, the low tide level must be equal or less than 10 cm higher than the low tide level registered at deployment. 3.0 Method for deployment (per environmental site) In the laboratory, on deployment day 1. Fill the 100-ml plastic containers, each with approximately 90 ml of RSW at 33-g/L salinity (although only three containers will be used at deployment, four are prepared for security reasons). 2. Transfer 10 juveniles of E. marinus to each of the 100-ml plastic containers, using the 3-ml plastic pipette with the tip cut. 3. Place the 100-ml plastic containers inside the thermally insulated box, ready for transportation to the field. Minimum disturbance is desirable during transportation. At the environmental site 4. Push each chamber into the sediment to a depth of about 14 cm and up to 1 cm under the top of the meshed windows (use the rubber gloves and boots). Deployments should be carried out at the lowest level of low tide and near the water line to avoid excessive desiccation of the sediment during the assay. 5. Release 10 organisms at the sediment surface of each assay chamber, by gently pouring the content of each 100-ml plastic container. 6. Introduce a small piece of local Fucus sp. to provide shelter and simulate its natural habitat (Marques and Nogueira, 1991).

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7. Cover the assay chambers immediately with the cup (see Figure 1, Appendix). 8. Expose the organisms in the field for 48 hours. 4.0 Method for assay termination (per environmental site) At the environmental site 1. Pull one assay chamber with one hand, and with the other hand close the bottom aperture with a cork (use the rubber gloves and boots). Pulling out the assay chamber should be done carefully to avoid loss of sediment from the assay chamber. 2. Place the assay chamber in a 1-L plastic bag previously tagged. 3. Repeat steps 1 and 2 for the other two replicates. 4. Fill the 5-L plastic bottles with site water. 5. Collect a sediment sample from the first 5 cm (approximately 1000 g wet weight) and place it in double 15-L plastic bag, to prevent breakage and loss of sample. 6. Place the 1-L plastic bags (containing the assay chambers) and the 5-L plastic bottles (containing the site water) inside the thermally insulated boxes, ready for transportation to the laboratory. 5.0 Method for postexposure feeding quantification (per environmental site) In the laboratory, after arriving from the field (define a fixed period between retrieval from the field and the beginning of the laboratory procedures for postexposure feeding quantification, to provide similar conditions across sites). 1. Unfreeze the 1.5-ml microtubes containing the Artemia nauplii. 2. Fill each of the 30-ml glass beakers with 5 ml RSW at 33-g/L salinity using the 5-ml micropipette. 3. Transfer the content of each 1.5-ml microtube to each 30-ml glass beakers using the Pasteur pipette. 4. Fill half the three petri dishes and the three 500-ml plastic containers with site water. 5. Take a 1-L plastic bag containing an assay chamber out of the thermally insulated box. 6. Rinse the surface of the assay chamber with the wash-bottle (filled with site water) in a plastic tray. 7. Collect the organisms (from each replicate) using the 3-ml plastic pipette with the tip cut and place first in a petri dish (to clean organisms of adherent particles with RSW) and later in a 500-ml plastic container 8. Transfer each organism from a 500-ml plastic container into each of seven 30-ml glass beakers using the 3-ml plastic pipette. 9. Place the 30-ml glass beakers at 20 °C in the dark and keep them under these conditions for thirty minutes, using the clock (the postexposure feeding period). 10. Repeat step 4 through 9 for the other two replicates. 11. At the end of the postexposure feeding period, remove the organism from the 30-ml glass beaker using the 3-ml plastic pipette and place it in the upper plate of a 6-cm petri dish previously labelled. 12. Look for nauplii that can be adherent to the body of the organism or medium pipetted, under the stereomicroscope, and annotate. 13. Transfer the remaining Artemia nauplii from the 30-ml glass beakers into the bottom plate of the same petri dish previously used in the step 11. 14. Clean the 30-ml glass beaker with RSW at 33-g/L salinity using the 3-ml plastic pipette and transfer the medium into the petri dish. 15. Count the nauplii remaining on the petri dish while discarding them with the help of the micropipette with bulb under the stereomicroscope (preferably with a black background). 6.0 Method for sediment characterization (per environmental site) 1. Take the plastic bag containing the sediment sample. 2. Homogenize the sample by removing visible indigenous animals and large debris with a forceps. 3. Place half of the sample in a tray or box, previously labelled.

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4. Dry the sample at room temperature, removing periodically, for some weeks until constant weight. Annotate the dry weigh (initial total dry weight). 5. Hydrated and sieved the sample in wet through a sieve of 63-µm mesh size to separate the clay material. 6. Dry the fraction retained by the 63-µm sieve in an oven at 50 ºC until constant weight. Annotate the dry weight weighted (to obtain the weight of the fraction < 63 µm). 7. Sieve the dry sample obtained in step 6 using the sieve mechanical shaker with a set of six sieves (63, 125, 250, 500, 1000, and 2000 µm), during 15 min with 1 mm of amplitude of movement and an interval of agitation of 30 sec. 8. Weight the sediment remaining on each sieve (including that sieved again through the 63 µm mesh). 9. Express the weights as percentage of the initial total dry weight. 7.0 Method for determining postexposure feeding rates (per environmental site) 1. Calculate the observed postexposure feeding rate (oPEF) of each organism using the following equation: oPEF = ni – nf where: ni = initial number of nauplii supplied. nf = final number of nauplii consumed in 30 min. Units as nauplii/amphipod/30 min. 8.0 Method for determining adjusted postexposure feeding rates Postexposure feeding of E. marinus is significantly influenced by the proportion of sediment with size between 500-1000 µm and 1000-2000 µm during exposure (Martinez-Haro et al., 2016). The adjustment of postexposure feeding rates is necessary to compare postexposure feeding rates across environmental sites presenting differences in sediment particle proportions. 1. Calculate the predicted postexposure feeding rate (pPEF) for each environmental site based on the proportion of sediment with size between 500-1000 µm and 1000-2000 µm, using the following equation: Arcsine pPEFi = 45.586 (± 2.012) – 1.059 (± 0.207)·X1i+ 1.871 (± 1.190)·X2i where: X1i = proportion of sediment with size between 500-1000 µm at the i environmental site. X2i = proportion of sediment with size between 1000-2000 µm at the i environmental site. 2. Calculate the pPEF for the arithmetic mean of each covariate for all environmental sites (pPEF’), using the following equation: Arcsine pPEF’ = 45.586 (± 2.012) – 1.059 (± 0.207)· 𝑋1 + 1.871 (± 1.190)·𝑋2 where: 𝑋1 = arithmetic mean of the proportion of sediment with size between 500-1000 µm at all environmental sites. 𝑋2 = arithmetic mean of the proportion of sediment with size between 1000-2000 µm at all environmental sites. 3. Calculate the adjusted postexposure feeding rate (adPEF) by correcting for the proportion of sediment with size between 500-1000 µm and 1000-2000 µm, using the following equation: adPEF = (oPEF · pPEF’) / pPEF

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APPENDIX

Figure 1. Design of in situ assay chambers (given by N. Dias, LIP - Laboratory of Instrumentation and Experimental Particles Physics - Coimbra). Note: ACRIFIX® 2R 0190 and ACRIFIX® CA 0020 was used for fixing nylon meshes to the lateral openings and cups.