Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Ethics statement: Protocols for assessing endocrine disrupting activity of chemicals/mixtures in fish have been approved by Brunel University London's Animal Welfare and Ethical Review Body (AWERB) and by the UK Home Office under the Animals (Scientific Procedures) Act 1986.

1. Water Sample Collection, Preservation and Extraction

1. Sample collection and preservation
   1. Prior to use, clean the bottles with a suitable surface active cleaning agent. After cleaning, rinse the bottles with water, drain and dry.
   2. Collect samples in glass bottles of 2-L capacity containing a preservative consisting of 0.5 g of copper(II) nitrate and 6 ml of 3.6 M hydrochloric acid solution. Store samples below 10 °C. Extract and analyze as soon as possible following collection and preservation.
2. Sample extraction and clean-up (for steroid estrogen analysis)¹⁰
   1. Solid Phase Extraction (SPE)
      1. Prior to extraction, to reduce suspended solids, filter water samples using 1 µm pore size filter paper.
         1. Once filtered, spike samples with internal standard by adding 100 µl of spiking deuterated internal standard stock solution containing 2,4,16,16-d₄-estrone: 2,4,16,16-d₄-17β-estradiol (E2): and 2,4,16,16-d₄-17α-ethinylestradiol (EE2) (all at 40 µg/L in methanol) to 1,000 ml effluent (or 100 ml influent) resulting in internal spike of 2 ng/L for sewage effluent samples, and 20 ng/L for sewage influent samples.
      2. Attach disposable valve liners, styrene divinyl benzene SPE cartridges, and cartridge reservoirs to the SPE apparatus. Switch on vacuum pump to test the equipment is adequately sealed.
      3. Pipette 5 ml of ethyl acetate into each reservoir, to condition the cartridges. Switch on the vacuum pump (to below 10 inHg to allow a flow rate of less than 10 ml per minute) and pull through the liquid. Do not let the SPE cartridges dry out. Repeat process with 5 ml of methanol followed by 5 ml of water.
      4. Top up each cartridge reservoir with water and connect 1/8" PTFE tubes between the cartridge reservoirs and glass sample bottles. Switch on the vacuum at a flow rate of less than 10 ml per minute and allow the whole sample to pass through the cartridge. Empty the waste flask as necessary.
      5. Thoroughly dry the SPE cartridges under vacuum (or by using air or nitrogen) until the cartridge contents change color (e.g., from dark brown to light brown).
      6. Put clean dry 10 ml glass collection vials into rack and place inside the extraction manifold. Check that each liner is above a vial. Pipette 8 ml of dichloromethane into each sample reservoir, switch on the vacuum pump (flow rate of less than 10 ml per minute) and pull the liquid through into the collection vials.
      7. Remove 10 ml vials from the SPE manifold and use a concentrator to reduce the volume to 1 ml. Transfer each sample into auto-sampler vial and further concentrate to 100 µl using nitrogen blow down equipment.
   2. Gel Permeation Chromatography (GPC) clean-up
      1. Inject 95 µl of the eluted sample extract into the GPC equipped HPLC using the conditions described in Table 1. Concentrate the GPC extract down to 200 µl using a concentrator and nitrogen blow down apparatus and then make up to 2.0 ml with hexane.
   3. SPE clean-up
      1. Attach disposable valve liners, aminopropyl cartridges, and cartridge reservoirs to the SPE manifold. Put clean dry 10 ml glass collection vials into rack and place inside the extraction manifold. Pipette 2 ml of hexane into each reservoir, to condition the cartridges. Allow the liquid to pass through the cartridges.
      2. Pipette the GPC sample extract into the reservoir and again allow the liquid to pass through the cartridge. Collect the sample eluate in the vial and remove from the manifold. Do not discard eluate.
      3. Put a new clean dry 10 ml collection vial into rack and place inside extraction manifold. To wash the cartridge, add 2 ml of ethyl acetate in hexane (30% v/v) to the reservoir and pull the liquid through the cartridge. Repeat with a further 2 ml of ethyl acetate in hexane, discarding all washings.
      4. Place the original 10 ml collection vial (step 1.2.3.2) back in rack inside the extraction manifold. Pipette 2 ml of ethyl acetate in acetone (50% v/v) into the reservoir, switch on the vacuum pump (to below 2 inHg to allow a flow rate of less than 2 ml per minute) and pull the liquid through into the vial. Repeat the process with another 2 ml of ethyl acetate.
      5. Remove the sample vial and use a concentrator to reduce the extract volume to 1 ml. Transfer the sample into a smaller glass vial and use nitrogen blow down equipment, to evaporate the extract to incipient dryness.
      6. Add 100 µl of methanol and mix well. Transfer the extract to an auto-sampler vial (with 0.3 ml insert) and cap the vial. Analyze the samples using LCMS/MS (see section 2).

Table 1. Conditions and parameters for Gel Permeation Chromatography (GPC) clean-up of extracted wastewater samples. Table details GPC column, mobile phase, temperature, injection volume, and detector wavelength.

3. Sample extraction (for Yeast Estrogen Screen)
   1. Filter samples as detailed in section 1.2.1.1. Attach disposable valve liners, C18 SPE cartridges, and cartridge reservoirs to the SPE apparatus. Switch on the vacuum pump to test the equipment is adequately sealed.
   2. Pipette 5 ml methanol into each reservoir, to condition the C18 cartridges. Turn on vacuum (to about 5 inHg to allow a flow of about 5 ml per minute) and let the liquid run through, pausing for 1 min half way through. Do not let the cartridges run dry. Repeat process with either HPLC grade or double distilled water (ddH₂O). Again do not run dry.
   3. Extract water samples as described in section 1.2.1.4. Continue the vacuum for at least 30 min to thoroughly dry the cartridges.
   4. Place clean dry 10 ml collection vials into a rack in the extraction manifold. Check that each liner is above a vial. Add 5 ml of methanol to each reservoir. Turn on the vacuum (maximum flow of 5 ml/min) and allow the liquid to pass through the cartridge into the vial, pausing for 2 min half way through.
   5. Prior to analysis using the YES screen, reduce the extract to incipient dryness using nitrogen and reconstitute with 500-1,000 µl ethanol. Seal the lids of sample vials to avoid evaporation and keep at 4 °C (in a spark-free fridge).

2. Chemical Analysis Using LCMS/MS

1. Optimize the operating conditions of the LCMS/MS system using manufacturer's instructions.
2. Analyze calibration standard solutions, the sample extracts, blank and analytical quality control (AQC) samples using the liquid chromatography and mass spectrometry conditions, and monitor the ion transitions as detailed in Table 2. Determine the concentration of steroid estrogens in the sample extract using internal standards¹⁰.

Table 2. Details parameters and conditions for LCMS/MS analysis of steroid estrogens in wastewater extracts. Table gives sample injection volume and flow rate, mobile phase conditions and gradient.

3. Estrogenic Activity Using In Vitro Yeast Estrogen Screen (YES) Assay⁸

1. Prepare and store minimal medium and medium components as per Table 3 (a) para (g).

Table 3. Yeast Estrogen Screen assay; preparation and storage of minimal medium and medium components.

2. Preparation and storage of 10x concentrated yeast culture
   1. On day 1, prepare growth medium (as detailed in 3.4.1) and pour into a sterile conical flask. Add 125 µl of 10x concentrated yeast from cryogenic vial stored at -20 °C. Incubate the inoculated media at 28 °C for approximately 24 hr on an orbital shaker.
   2. On day 2, make two bottles of growth medium (~ 50 ml) and pour into separate sterile conical flasks. Add 1 ml of 24-hr cultured yeast into each flask of growth media. Incubate the inoculated media at 28 °C for approximately 24 hr on an orbital shaker.
   3. On day 3, pour each 24-hr culture into a sterile 50-ml centrifuge tube. Centrifuge the 50 ml tubes at 4 °C for 10 min at 2,000 x g. Pour off the supernatant, and re-suspend each pellet in 5 ml of minimal medium with 15% glycerol. Make 0.5 ml aliquots of the 10x concentrated yeast culture in 1.2-ml sterile cryovials and store at -20 °C for a maximum of 4 months.
3. Preparation and storage of chemical stocks for standard curves
   1. Rinse all glassware and spatulas twice with absolute ethanol and leave to dry before use to remove any traces of contaminates.
   2. Weigh E2 directly into a glass vial in a powder weighing cabinet and adjust concentration by volume in absolute ethanol. Dilute to a concentration of 2×10^-7 M (54.48 µg/L). Seal lid and store at 4 °C (in a spark-free fridge).
4. Assay producer
   1. On day 0, prepare growth medium. To the 45 ml bottle of minimal medium add 5 ml glucose solution, 1.25 ml L-aspartic acid solution, 0.5 ml vitamin solution, 0.4 ml L-threonine solution, and 125 µl copper(II) sulfate solution. Pour growth medium into a sterile conical flask.
      1. Add 125 µl of 10x concentrated stock (defrosted from -20 °C storage) to the flask and incubate the inoculated media at 28 °C for approximately 24 hr on an orbital shaker.
   2. On day 1, label a sterile 96-well 'dilution plate' and make serial dilutions (100 µl volumes in ethanol) of E2 standard curve and test chemical(s)/effluent extract(s) (e.g., EE2).
   3. Label sterile 96-well optically flat bottom microtiter 'assay plate(s)'. On every plate include blanks (plus/minus solvent) and an E2 standard curve, in addition to rows of test chemical(s)/effluent extract(s).
   4. Pipette 10 µl of each concentration (E2, chemical or extract) into the appropriate well of the assay plate. Pipette 10 µl of ethanol into each 'solvent blank' well of the assay plate. Leave assay plate with the lid off to evaporate to dryness.
   5. Make a bottle of growth medium (~ 50 ml) and add 0.5 ml of chlorophenol red-β-D-galactopyranoside (CPRG) solution per bottle. Determine the density of yeast cells in the 24-hr culture by measuring turbidity of the culture at 620 nm in a plate reader. Inoculate the assay medium with 4×10⁷ yeast cells from the 24 hr culture.
   6. Pour inoculated assay medium into a sterile trough. Using a multi-channel pipette add 200 µl of inoculated assay medium to each well of the 96-well assay plate.
   7. Put the lid on the 96-well assay plate and seal the edges with tape. Shake the assay plate(s) vigorously for 2 min on a titer plate shaker and incubate at 32 °C in a naturally ventilated heating cabinet.
   8. On day 2, shake the assay plate(s) vigorously on a titer plate shaker for 2 min. Return to 32 °C incubator.
   9. On day 4, shake the assay plate(s) vigorously for 2 min on a titer plate shaker. Leave the plate(s) to stand for approximately 1 hr then read the assay plate(s) at an absorbance of 540 nm (optimum absorbance for CPRG ~575 nm) and 620 nm (for turbidity) using a plate reader. Leave plate(s) at room temperature and read later if necessary.
5. Estrogenic activity calculations
   1. Correct assay readings for turbidity using the following equation: Corrected value = sample or standard (E2) absorbance at 540 nm – [sample or standard (E2) absorbance at 620 nm – blank absorbance at 620 nm]. Plot E2 standard curve with appropriate blanks (to check for contamination)⁸.
   2. Use the corrected sample (extract or chemical) to calculate 'Estradiol equivalents'. Use the plotted E2 standard curve values and regression equation (3-parameter polynomial or linear depending on fit) to interpolate the sample/extract absorbance values to E2 equivalent values. Use concentration factor (i.e., water extracted and volume of ethanol the extract is re-suspended in) to calculate estrogenic activity in pre-extracted sample.

4. Laboratory Based Assessment of Estrogenic Activity Using In Vivo Vitellogenin Induction in Male Fathead Minnows

1. Use male fathead minnows (Pimephales promelas) >4 months old, which display secondary sexual characteristics (i.e., the development of nuptial tubercles and a dorsal fatpad) indicative of male sexual determination.
   1. To prevent spawning activity, separate mature males three weeks (± 3 days) prior to the initiation of the test. Set up at least two 45 L glass aquaria with a maximum loading of 3 g/L. To ensure adequate numbers of healthy fish for the test, use a minimum of 100 males.
   2. Keep the male fish under identical environmental conditions as will be experienced in the test (i.e., water temperature of 25 ± 1 °C and a 16:8 hr light:dark photoperiod). Maintain the dilution water flow rate to each tank to ensure a 95% replacement time of at least every 6 to 8 hr, i.e., 330 ml/min for a 45 L tank.
2. Test apparatus and experimental design
   1. Use large glass tanks to accommodate a loading of up to 3 g of fish per liter of water. For 8 adult males (nominally 4.5 g each), use a 10-20 L tank.
      1. Use two replicate tanks for each treatment and shield each tank from any unnecessary visual disturbances (i.e., use laminated card screens between the tanks). Identify each tank with a study number, an exposure concentration and a vessel identification number.
   2. For wastewater effluent studies
      1. On arrival, immediately transfer effluent into a storage tank at 10 ± 1 °C. Start dosing effluent within two hr of receipt of the effluent.
      2. Feed the effluent, via peristaltic pump, from the 10 °C storage tank to an acclimation vessel. Heat the effluent in the acclimation vessel to 18 ± 2 °C. Pump the heated effluent from the acclimation vessel to the dosing/mixing vessels and then to the test aquaria (which holds the fish). Heat the aquaria to a temperature of 25 ± 1 °C.
   3. For chemical dosing studies
      1. Weigh test chemicals in a powder weighing cabinet. Prepare concentrated chemical stocks in ddH₂O preferably without the use of solvents.
         Note: If solvents are required to solubilize test compounds, use solvent controls in addition to the dilution water control.
      2. Gravity feed or pump dilution water from a temperature controlled header tank via flow control device(s) to dosing/mixing vessel. Pump concentrated chemical stock(s) using a peristaltic pumping system to the dosing/mixing vessels. Control the pump rate (of stock chemical) and water flow rate (dilution water) to achieve the desired exposure concentration.
         Note: Use silicone tubing to feed water/test chemical to each test vessel from the dosing/mixing vessel. Use a flow rate that is sufficient to provide a 75% vessel replacement in at least 24 hr, i.e., 20 ml/min for a 20 L tank. Maintain the flow rate at ± 10% of the specified nominal value.
   4. Maintain exposure tank water temperature at 25 ± 1 °C, dissolved oxygen above 70% of the air saturation value (5.8 mg L^-1 at 25 °C) and ± 0.5 pH units (starting pH between 6.5 and 8.5) throughout study. Set lighting conditions to photoperiod of 16 hr light: 8 hr dark, with dawn/dusk transition periods of 20 min.
   5. Feed the fish twice a day with freshly defrosted frozen adult brine shrimp (Artemia) at 2.5 (± 0.1) g per tank per feed. Also feed the fish once a day with a small quantity (two finger pinch) of a tropical flake fish food. Leave a minimum of 3 hr between each feed.
      1. Keep a daily feeding record to monitor the feeding response/behavior (good, moderate or poor, compared with the controls). Siphon the tanks at least twice a week (preferably daily) to remove any uneaten food and feces. Clean the sides and bottoms of the test vessels at least once per week.
   6. Take weekly water samples from each tank to confirm estrogenic activity (via Yeast screen) and chemical composition (via analytical chemistry). See sections 1-3 for details of water sampling, extraction and analysis.
      Note: For each experiment, use dilution water control (negative control), positive control (E2 or EE2) plus at least three dilutions of effluent (100, 50 and 25%) or test chemical to monitor dose response. If novel technologies are tested, use compound/effluent with or without treatment (e.g., EE2 without treatment, EE2 with TAML/hydrogen peroxide (H₂O₂) treatment¹²; Figure 1).

Figure 1. Diagram representing experimental design of an in vivo fathead minnow vitellogenin bioassay to determine removal of ecotoxicity of 17α-ethinylestradiol using TAML/peroxide water treatment. The experimental set up consists of eight 11 L glass aquaria each fed with continuous flow of water. Individual chemical stock solutions and water (filtered de-chlorinated) are delivered to the mixing chambers. Nominal concentrations (without reaction) in the mixing vessels are 2 ng/L EE2, 80 nM TAML and 0.16 µg/L H₂O₂. Chemical stock solutions (EE2, H₂O₂ and TAML) are prepared and dosed separately so that the reactions commence in the mixing vessels. Fish (8 male fathead minnows per tank) are exposed to the mixture(s) after a reaction contact time of approximately 45 minutes. Water samples are taken from the exposure tanks weekly. Plasma samples are taken from fathead minnows to measure the estrogenic biomarker vitellogenin (VTG) from a baseline group, at the start of the study, and all other fish after 21 days of exposure. The specific treatments are: '-C'; negative control (dilution water only), '+'; positive control of EE2, '+H'; EE2 plus H₂O₂, '+T'; EE2 plus H₂O₂ plus TAML. This figure has been modified from Mills et al. 2015¹². Please click here to view a larger version of this figure.

3. Test Procedure
   1. Acclimation period
      1. Measure wet weight (g) of each sexually mature male fish, and allocate at random to each tank (8 males per tank).
      2. Keep unallocated fish from the same batch and maintain them under the same test conditions. Use these fish to replace any individuals that show signs of physical damage or a lack of condition during the 7-day acclimation period. Ensure at the end of the acclimation period each treatment tank has 8 males that have fully acclimated to the test conditions.
      3. Take 'baseline' plasma samples from an additional 8 males from the same batch of male fish. Follow the blood sampling method in section 4.4 and the Vitellogenin (VTG) analysis method in 4.5.
   2. After the acclimation period, deliver effluent or chemical dosing stocks to the exposure tanks for 21 days as described in 4.2.2 or 4.2.3. Monitor mortality, behavior and physical appearance of the fish in each replicate tank daily prior to the first feed of the day. Record any abnormal behavior or incidents.
      Note: Ensure environmental conditions and feeding rates maintain the health of the fish (sections 4.2.4 and 4.2.5).
4. Fish Sampling after 21-day exposure period
   1. 12 hr prior to sampling, stop feeding the fish.
   2. Label micro-centrifuge tubes for the collection of blood samples, add ~5 µl of Aprotinin (a protease inhibitor) and place them on ice.
   3. Make a 500 mg/L solution of MS222 (anesthetic) by dissolving 500 mg of MS222 per 1 L of de-chlorinated water (previously acclimated to 25 ± 1 °C). Neutralize the MS222 to pH 7.4 ± 0.4 using 1 M NaOH.
   4. Move each fish from the tank into the buffered MS222. Keep in the solution until the cessation of all operculum movement (typically 5 ± 1 min).
   5. Measure and record the fork length (mm) under the terminal anesthetic. Use a disposable scalpel to amputate the tail, and use a heparinized hemocrit tube to collect the blood from the caudal artery (Figure 2). Carefully dispense the blood into the pre-labeled microcentrifuge tube and keep on ice.
   6. Kill the fish immediately after drawing the blood. Confirm death by permanent cessation of the circulation and/or the destruction of the brain. Measure and record total fish weight (to nearest 0.01 g), and dissect tissues as required.
   7. Centrifuge the blood (7,000 x g for 5 min at 4 °C) within 2 hr of collection. Transfer the plasma supernatant using a pipette into new labeled 0.4 ml microcentrifuge tubes and store on ice. Freeze the tubes containing plasma on dry ice within 30 min of centrifugation and store at -80 °C before analysis of plasma VTG concentrations.

Figure 2. Photos depicting male fathead minnow (Pimephales promelas), plasma collection and location of testis. At the end of the 21-day exposure all fish should be killed to collect blood samples. Once under this terminal anesthetic fish length (fork length, mm) should be measured, quickly followed by blood collection from the caudal artery. Photo-A: red dotted line indicates location for tail amputation (a disposable scalpel should be used to amputate the tail). Photo-B shows a heparinized hemocrit tube used to collect the blood. Each fish should then be killed immediately after its blood sample has been taken, in this case the whole head has been severed from the body (Photo-C). Once the fish has been killed the body cavity can be opened up to reveal the internal organs. Photo-C shows the location of the testis (gonad) in relation to the swim bladder (SB) in cyprinid fish, e.g., fathead minnow, roach, carp, etc. Please click here to view a larger version of this figure.

5. Measure plasma VTG concentrations with a homologous VTG enzyme-linked immunosorbent assay (ELISA) kit designed specifically for fathead minnows.
   1. Prepare VTG standards and buffers as outlined in the manufacturer's protocol. Dilute plasma sample 1:50, 1:5,000, and 1:500,000 and assay them in duplicate to obtain readings within the VTG standard curve range as per manufacturer's instructions. Follow manufacturer's guidelines to calculate vitellogenin concentrations.

5. Field Assessments of Advanced/Novel Wastewater Treatment Technologies to Mitigate Estrogenic Activity Using In Vivo Vitellogenin and Intersex Induction in Roach (Rutilus rutilus)

1. Capture of wild roach living down stream of Wastewater Treatment Plant outfalls
   Note: Use roach (Rutilus rutilus) or other abundant freshwater or brackish fish species, which are gonochoristic and known to be sensitive to estrogenic endocrine disruption.
   1. Catch fish using electrofishing, netting, trapping or other recognized fishing methods depending on the situation¹³. Transport the fish in aerated tanks back to laboratory for sampling.
   2. Prepare microcentrifuge tubes as detailed in 4.4.2. Prepare buffered MS222 as described in 4.4.3. Anesthetize fish as detailed in 4.4.4.
   3. Measure fish length and weight under terminal anesthetic.
   4. Collect blood from the caudal artery using disposable heparinized syringe. Kill each fish immediately after blood sample collection. Carefully dispense the blood into pre-labeled microcentrifuge tubes and keep on ice. Prepare plasma as described in step 4.4.7 and measure VTG by ELISA (4.5).
   5. Using forceps remove 2-3 fish scales from each fish and place in individually labeled small paper envelopes. Store envelopes at room temperature in dry conditions for later fish age determination and growth analysis.
   6. Open body cavity with a scalpel to reveal the internal organs. Take out the gut to reveal the swim bladder with a gonad located on either side (Figure 2). Carefully remove the paired gonads with fine nosed forceps and place into a glass vial. Cover the gonads with Bouin's fixative at a ratio of 1:10 tissue: fixative.
   7. Leave the tissue in Bouin's for 6-24 hr depending on the size of the tissue (fixative penetrates at 1 mm per hr). Once fixed, pour off the Bouin's fixative and replace with 70% industrial methylated spirits (IMS). Store the fixed tissue at room temperature until tissues are processed for histopathology (section 5.3).
2. Field based assessment of estrogenic activity using in vivo vitellogenin and intersex induction in roach
   1. Experimental design and set up
      Note: Set up the tanks and pilot plant well in advance of the in vivo work starting. Start the tanks flowing 3-4 weeks prior to any addition of fish to the system. Monitor water flow rates, water quality and conditions (pH, temperature, dissolved oxygen, etc.) regularly to make sure parameters can be maintained.
      1. Construct large tanks (e.g., 300-1,000 L) on site at the pilot plant, which can receive 'control' de-chlorinated tap water, standard treated effluent(s) and advanced treated effluent(s) in parallel. Attach air pump/aerators to the tanks. Set water flow rates to achieve at least 6 tank volume exchanges per day.
         Note: Make sure tanks are well insulated and shaded to prevent excessive daily temperature fluctuations. Design tanks to allow observation of the fish, for behavioral alterations (lack of feeding, etc.), signs of disease and mortality.
   2. Start the exposure by floating the bags of roach (from the fish farm) in the respective tanks for 1 hr. Add tank water to the bags gradually until water temperature and conditions are ambient. Release the fish into their tanks.
   3. Feed adult roach daily on pelleted fish food (size 0.5−0.8). Feed juvenile roach daily on small pelleted (pellet sizes 100−300) non-estrogenic feed¹⁴ ad libitum, adjusted on the basis of uneaten feed. Keep a daily feeding record documenting feeding response/behavior (good, moderate or poor, compared with the controls).
   4. Take weekly water samples from each tank to confirm estrogenic activity and chemical composition. See sections 1-3 for details of water sampling and analysis methods.
3. Histopathology of fish gonads¹⁵
   1. Carefully remove the dissected and fixed gonads (described in steps 5.1.6 and 5.1.7) from the container with tweezers and place on a cutting board.
   2. Use a microtome blade to cut each gonad into 3 parts (anterior, mid and posterior) and from each part cut a 3-5 mm thick cross section. Carefully place all six pieces into a labeled plastic biopsy cassette, and place into tissue processor using the timings listed in Table 4.
   3. Wax embed tissues and section on rotary microtome (3-5 μm). Transfer sections to labeled bio-adhesive coated glass slides and place slides on a heated plate (set at 45 °C) to dry for 24 hr.
   4. Stain the slides, either manually or using an automated stainer, using the timings detailed in Table 5. Place a drop of mounting agent on the stained tissue, and lay a glass coverslip over the mounting agent to protect the tissue.
   5. First, examine each slide at low magnification (20X, i.e. 2X objective, with 10X eye lines magnification) to determine sex and the number of points of attachments to the body cavity¹⁵. Note any abnormalities for each fish.
   6. At a higher magnification (100X or 400X), examine the tissue to assess gametogenesis stages, abnormalities and the presence of oocytes in testicular tissue. Record the severity of intersex using the following grading system ranging from 0 (normal male tissue) to 7 (100% ovarian tissue)⁶ (see Table 6).

Table 4. Processing regime for wax impregnating tissues for histopathology. Tissues should be processed in an automatic tissue processor. Tissues should be immersed in the solutions detailed for the period of time specified.

Table 5. Solutions and immersion times for Haematoxylin and Eosin (H & E) staining of fish gonadal tissues. Slides should be placed in each bath for the allocated time in sequence. H & E staining of tissues is required to determine developmental or organizational impacts of estrogenic wastewater effluents on fish gonads.

Table 6. Scoring system to assess severity of intersex condition in roach. Histologically prepared slides of gonadal tissue should be examined under light microscope, at 20X, 100X and 400X magnification, to assess any abnormalities and the presence of oocytes in testicular tissue. This table is modified from Jobling et al. 2006⁶.