Canadian add-on procedure to ISO 10710: Test for determining growth inhibition using the macroalgae, Ceramium tenuicorne

Method Development and Applications Unit
Biological Assessment and Standardization Section
Science and Technology Branch
Environment and Climate Change Canada

Report STB 1/RM/63

March 2025

List of figures

Figure 1 Healthy female gametophytes of Ceramium tenuicorne (marine clone) upon receipt from the University of Oslo (AquaTox, 2022)

Figure 2 Example of Ceramium tenuicorne used to prepare new culture vessels

Figure 3 Examples of C. tenuicorne female gametophyte tips, 0.6–1.2 mm in length, that are suitable for beginning the growth inhibition test (AquaTox, 2022)

Figure 4 C. tenuicorne female gametophyte with one claw instead of two (AquaTox, 2022)

Figure 5 Examples of healthy and unhealthy Ceramium tenuicorne at test end (AquaTox, 2022)

List of tables

Table 1 Checklist of required and recommended culturing conditions for Ceramium tenuicorne for the purposes of Canadian-regulated sublethal toxicity testing

Table 2 Checklist of required and recommended test conditions and procedures for toxicity tests using Ceramium tenuicorne for the purposes of Canadian-regulated sublethal toxicity testing

Acknowledgements

This procedural document was prepared by Carolyn Martinko and Leana Van der Vliet (Method Development and Applications Unit, Environment and Climate Change Canada, Ottawa, ON), under the direction of Rick Scroggins (retired, Environment and Climate Change Canada, Ottawa, ON). We acknowledge Sarah Costantini, Melanie Gallant, Lisa Taylor, and technical staff from Nautilus Environmental (previously AquaTox Testing & Consulting Inc., Puslinch, ON), notably: Paige Cochrane, Nadia Pajnic, Anna Sobaszek, Cintia Glasner Regis, and Vanessa Chanes, for their contributions to adapting the ISO 10710 standard for use in Canadian laboratories. Barb Buckland (retired, Environment and Climate Change Canada, Gatineau, Quebec) is thanked for reviewing early drafts of this add-on procedure. Lindsey Boyd, Hana Moidu, and François Boisvert (Environment and Climate Change Canada, Gatineau, Quebec) are thanked for providing support and funding throughout method validation. The assistance of the late Dr. Stein Fredriksen (Department of Biosciences, University of Oslo, Oslo, Norway), who provided technical advice and supplied the algae culture used for method validation, is gratefully acknowledged. Kerry Dykens and Mark Hurd (National Center for Marine Algae and Microbiota [NCMA], Bigelow Laboratory for Ocean Sciences, East Boothbay, Maine), who accepted a deposit of the algae culture to the NCMA public collection and provided technical advice, are gratefully acknowledged.

1.0 Application

This Canadian add-on procedure for culturing and testing with Ceramium tenuicorne must be used when performing sublethal toxicity testing for facilities complying with Canadian regulatory requirements, and is applicable for effluents discharged to a marine or estuarine environment.

This add-on procedure must be used in conjunction with the International Organization for Standardization (ISO) standard 10710 (ISO, 2010), a 7-day static growth inhibition test using the red macroalga, Ceramium tenuicorne. ISO standards are available for purchase through several organizations, including directly from ISO www.iso.org. Note that all ISO standards are under copyright protection. The conditions and procedures described in this document include certain instructions that differ from the guidance provided in ISO (2010). The culturing and testing conditions in this add-on procedure have been validated in a Canadian laboratory through conducting tests with a reference toxicant chemical as well as with numerous pulp and paper mill effluents of different processing types, and with several metal mining effluents (AquaTox 2022, 2023; Nautilus Environmental, 2024). The test was consistently found to be robust, reliable, and sensitive in response to diverse effluent types.

In this document, as in other Environment and Climate Change Canada (ECCC) standardized toxicity test methods, the following levels of procedural stringency apply:

• “must” is used to express an absolute requirement;
• “should” is used to state that the specified condition or procedure is recommended and ought to be met if possible;
• “may” is used to mean “is (are) allowed to”;
• “can” is used to mean “is (are) able to”; and
• “might” is used to express the possibility that something could exist or happen.

Note that the use of “shall” and the present tense “is” or “are” in an ISO standard is equivalent to a “must” or “requirement” in an ECCC method. The use of “should” in an ISO standard is interpreted in the same way as in an ECCC method; it is equivalent to “recommended.” In the ISO (2010) standard, commas are used in place of decimals in measurements (i.e., 6,5 is equivalent to 6.5).

2.0 Rationale

Several effluent regulations pursuant to the Fisheries Act specify the use of marine macroalgae (i.e., seaweed) test methods as part of a test battery to monitor effluent quality. The specified test methods are those standardized and published by the United States Environmental Protection Agency (USEPA) and can use either of two test species: Macrocystis pyrifera or Champia parvula. Historically, tests using Champia parvula have been successfully completed for East Coast facilities. However, over the past several years, there have been significant issues with the reproducibility of the test using this species, leading to concerns about data quality. The only laboratory in North America that conducted tests using Champia parvula has discontinued this service, resulting in a lack in testing capacity. Tests using Macrocystis pyrifera are being successfully completed for West Coast facilities and recently for East Coast facilities. However, Macrocystis pyrifera may not be representative of East Coast fauna, and there are significant logistical challenges involved in acquiring both the test species and effluent within the timeframe required by the test method.

In order to restore the previous flexibility in test species options, ECCC investigated other standardized test methods using marine macroalgae species and focused on the ISO 10710 standard method for Ceramium tenuicorne. After confirming that Ceramium tenuicorne is ecologically relevant to the Canadian marine environment (Garbary and McDonald, 1996; Sloan and Bartier, 2000; ECCC, 2020), ECCC’s Method Development and Applications Unit (MDAU) reviewed the ISO (2010) standard and developed the additional procedures and conditions necessary to ensure compatibility with other sublethal toxicity test methods specified in Canadian regulations. This add-on procedure for conducting the ISO 10710 standard method in a Canadian context was further refined through research conducted at a Canadian commercial laboratory (AquaTox, 2022, 2023; Nautilus Environmental, 2024). As stated in Section 1, this add-on procedure must be used in conjunction with the ISO 10710 standard (ISO, 2010).

3.0 Test organisms

3.1 Species and source

The red macroalgae, Ceramium tenuicorne ([Kützing] Wærn, 1952; division Rhodophyta, order Ceramiales), is to be used as the test species. Dixon (1960) and Cho et al. (2001) describe the structure of the genus Ceramium. Due to its structure, each cell of Ceramium tenuicorne is in direct contact with its surrounding aquatic environment and is likely to be affected by soluble toxicants (Eklund, 1998). Two clones isolated from different habitats were originally considered to be distinct species; however, DNA sequencing and interfertility studies concluded that these organisms belong to the same species (Rueness, 1978; Gabrielsen et al., 2003). The marine clone (previously known as Ceramium strictum) originated from the Oslofjord (20–25 parts per thousand [‰] salinity), while the brackish water clone originated from the Baltic Sea, south of Stockholm (7‰ salinity) (Rueness, 1973; Eklund, 2005). The ISO (2010) method has been successfully used for both clones. In Canada, the marine clone of C. tenuicorne (C. strictum) has been found in Monks Head Harbour, Nova Scotia (Garbary and McDonald, 1996) and the Haida Gwaii archipelago (Queen Charlotte Islands), British Columbia (Sloan and Bartier, 2000).

This add-on procedure must be performed using female gametophytes of the marine clone of Ceramium tenuicorne, as females grow faster than male gametophytes and grow more uniformly in a dichotomous pattern than diploid tetrasporophytes (Bruno and Eklund, 2003; ISO, 2010), and the marine clone can be acclimated to and cultured at 30‰ salinity (Eklund, 2005; Macken et al., 2012). Female gametophytes of the marine clone of Ceramium tenuicorne sourced from the University of Oslo were used throughout all stages of validating this add-on procedure (AquaTox 2022, 2023; Nautilus Environmental, 2024). Taxonomic identification and documentation of the test organism species, clone, and sex must be completed; this requirement can be met by certification from the source. See Appendix D for more details on structure and distinguishing female and male gametophytes.

Ceramium tenuicorne is available for purchase from the following supplier:

National Center for Marine Algae and Microbiota (NCMA), Bigelow Laboratory for Ocean Sciences
60 Bigelow Drive, East Boothbay, ME 04544
Strain: Ceramium tenuicorne (Kützing) Waern
Phone: (207) 315-2567 ext. 3
Email: ncma@bigelow.org

For current information on suppliers for C. tenuicorne, contact:

Method Development and Applications Unit
Science and Technology Branch
Environment and Climate Change Canada
335 River Road
Ottawa, ON K1V 1C7
Email: methods@ec.gc.ca

Figure 1 shows examples of healthy female gametophytes of the marine clone of C. tenuicorne received in a laboratory from an established source. Appendix D provides additional details on culture health.

3.2 Acclimation

Acclimation of C. tenuicorne to laboratory culturing conditions (e.g., media, temperature, light intensity, photoperiod) should be as gradual as possible to avoid stressing the organisms (EC, 1999; AquaTox, 2022). C. tenuicorne cultures must be acclimated to a salinity of 30 ± 2‰, which is the salinity used for sublethal toxicity testing of effluents discharged to a marine or estuarine environment, as required by Environment Canada guidance (EC, 2001). Acclimation of C. tenuicorne cultures to this salinity should be gradual, with a change of ± 3‰ salinity every other day until the desired salinity is reached (Section 5 of ISO, 2010). C. tenuicorne cultures must be acclimated to the salinity and temperature used for testing for at least two weeks prior to beginning a test (ISO, 2010).

3.3 Culture conditions

Table 1 provides a summary of the required and recommended conditions for culturing Ceramium tenuicorne to obtain organisms for Canadian-regulated sublethal toxicity testing. The conditions described in this section supplement and in some cases supersede the specifications in ISO (2010).

Section 6 of ISO (2010) describes apparatus recommended for conducting culturing and testing. Ceramium tenuicorne should be cultured and maintained in a facility where temperature and lighting can be controlled. The culturing area should be isolated from any testing or sample preparation areas to reduce the possibility of culture contamination by test substances or materials. Equipment that comes in contact with C. tenuicorne or media must be made of non-toxic, chemically inert material; equipment made of copper must not be used (Section 4.2.1 of ISO, 2010).

Aseptic technique should be used for culturing C. tenuicorne.^{Footnote 1} Cultures of C. tenuicorne must remain free of other algae and blue-green algae (i.e., cyanobacteria); however, completely axenic cultures are not necessary (S. Fredriksen, University of Oslo, personal communication, 2020; AquaTox, 2023).

C. tenuicorne that are to be used for tests involving wastewater must be cultured in media prepared according to Section 3.4 of this add-on procedure. Organisms must be transferred to fresh culture media weekly in order to maintain actively growing female gametophytes (Section 5 of ISO, 2010).^{Footnote 2} The cultures are not aerated. The culture vessels should be sterile plastic or glass Petri dishes with lids; the recommended size is 90 × 15 mm or 100 × 15 mm.^{Footnote 3} The recommended loading density is approximately 20–30 algae tips that are each 10 to 20 mm in length, per 25 mL of sterile culture media in each vessel (ISO, 2010). Algae tips can be cut to the appropriate size using the equipment and techniques described in Section 4.3 of this add-on procedure (i.e., using a scalpel or miniature scissors, tweezers, stereomicroscope and/or magnifying glass, and graph paper). Figure 2 shows examples of algae tips used for culture renewal. Algae tips used for culture renewal should have one branching point; however, algae tips that do not have a branching point but appear otherwise healthy (i.e., red colour, normal appearance) may be used for culture renewal if there is a limited number of organisms available (Nautilus Environmental, 2024).

Table 1: Checklist of required and recommended culturing conditions for Ceramium tenuicorne for the purposes of Canadian-regulated sublethal toxicity testing

Species

• - Ceramium tenuicorne female gametophytes, marine clone; species, clone, and sex confirmed

Culture medium

• - uncontaminated natural seawater, filtered through a paper filter (30 µm) and sterilized by autoclave or sterile filtration (0.2 µm) before use
• - nutrient solutions added according to Column A of Table 3 in ISO (2010)
• - salinity must be 30 ± 5‰ and should be 30 ± 2‰

Culturing vessel

• - sterile polystyrene or glass Petri dish with lid
• - 90 × 15 mm or 100 × 15 mm

Volume

• - approximately 25 mL of sterile culture media per vessel

Loading density

• - approximately 20 to 30 algae tips, each 10 to 20 mm in length, per 25 mL

Maintenance

• - subcultured weekly in fresh sterile culture media
• - temperature, salinity, pH, and dissolved oxygen monitored regularly (e.g., at the time of media renewals)
• - light intensity monitored weekly, as a minimum

Temperature

• - 22 ± 2 °C

pH

• - 8.0 ± 0.2, adjusted before and after sterilization if necessary using 1 mol/L HCl or 1 mol/L NaOH

Dissolved oxygen

• - 90 to 100% saturation; culture media aerated to within this range before use if necessary

Aeration

• - culture vessels are not aerated

Lighting

• - “daylight” or “warm white” fluorescent or equivalent light
• - intensity of 35 ± 7 μmol m^-2 s^-1
• - photoperiod of 14 h light : 10 h dark

Health criteria

• - culture must appear healthy and maintain an acceptable level of sensitivity, as determined through reference toxicant testing and warning charts

The information in this table is for summary purposes only. Definitive requirements and recommendations of this add-on procedure are contained in the main body of this document.

C. tenuicorne should be cultured at a temperature of 22 ± 2 °C. If temperature in the culture vessels (or in one or two extra vessels set up for the purpose of monitoring water temperature) is based on measurements other than those in the vessels themselves (e.g., in the incubator or controlled temperature room within the vicinity of the culture vessels) the relationship between the readings and the temperature within the culture vessels must be established and periodically checked to ensure that the algae are being cultured within the desired temperature range.

C. tenuicorne should be cultured using “daylight” or “warm white” fluorescent or equivalent lighting. The light fluence rate (intensity) should be 35 ± 7 µmol m^-2 s^-1 as measured at points approximately the same distance from the light source as the test organisms. Quantum methods must be used to measure light, as these methods are appropriate for photosynthetic organisms.^{Footnote 4}

The photoperiod should be 14 h light : 10 h dark. Light intensity should be measured on a weekly basis (AquaTox, 2022), as a minimum.

The culture of C. tenuicorne female gametophytes to be used for testing must appear healthy and maintain an acceptable level of sensitivity, as determined through reference toxicity testing and warning charts (see Section 4.8 of this add-on procedure). Figure 5 in Section 4.5 depicts examples of healthy and unhealthy characteristics of C. tenuicorne observed at the end of a reference toxicant test. Also see Appendix D for descriptions of sex identification, normal colouration and discolouration, and contamination of C. tenuicorne. Contaminated C. tenuicorne cultures (e.g., with microalgae, protozoa, fungi, or bacteria) should be discarded.

3.4 Culture media and water quality

Water used to prepare culture media must be an uncontaminated supply of natural seawater; its salinity must be 30 ± 5‰ and should be 30 ± 2‰.^{Footnote 5} At this time, there is no data available to support the use of artificial seawater in culturing.^{Footnote 6} See Section 4.2.3 in ISO (2010). Uncontaminated natural seawater must be filtered (e.g., 30 µm paper filter) to remove large particles. The pH must then be checked; if necessary, the pH must be adjusted to 8.0 ± 0.2 using 1 mol/L HCl or 1 mol/L NaOH.^{Footnote 7} After any pH adjustment, the natural seawater must be sterilized by autoclave or sterile filtration (0.2 µm pore size) before use. The pH of the natural seawater must be checked after sterilization, and if necessary the pH must be re-adjusted to 8.0 ± 0.2 using 1 mol/L HCl or 1 mol/L NaOH before use. Nutrient stock solutions must be prepared according to Section 4.3 of ISO (2010).^{Footnote 8} Nutrient solutions (i.e., nitrogen, phosphorus, iron, trace element, and vitamin solutions) must be added to the sterilized, pH-adjusted (if necessary) natural seawater according to the concentrations listed in Column A of Table 3 in ISO (2010). The dissolved oxygen content of this prepared culture media should be 90 to 100% saturation before use. If necessary to achieve that, mild aeration of the media should be carried out using filtered, oil-free compressed air; such aeration also assists in mixing the media. Overly vigorous aeration should be avoided (EC, 2011).

Culturing conditions including temperature, salinity, pH, and dissolved oxygen must be monitored and assessed at regular intervals (e.g., at the time of culture media renewal). Assessment of other variables such as total dissolved gases, ammonia, nitrogen, nitrite, metals, pesticides, suspended solids, and total organic carbon should be performed as frequently as necessary to document water quality.

4.0 Test system and procedure

The purpose of this test is to measure the sublethal toxicity of Canadian industrial wastewater effluent discharged into marine or estuarine environments on a species of macroalgae. Table 2 provides a summary of the required and recommended test conditions and procedures for conducting toxicity tests on effluents or reference toxicant chemicals using Ceramium tenuicorne for the purposes of Canadian-regulated sublethal toxicity testing. This guidance supplements and in some cases supersedes specifications in ISO (2010).

4.1 Sample collection, labelling, transport, and storage

Sample volume requirements depend on the number of test concentrations and the number of replicates. Generally, a 1-L sample of effluent is sufficient for conducting a test for algal growth inhibition. The sample must be collected and placed in a labelled or coded container made of non-toxic, inert material. Labelling or coding should include at least sample type, source, date and time of collection, and name of sampler(s). The container must be new or thoroughly cleaned, and rinsed with uncontaminated water. It should also be rinsed with the sample to be collected, and then filled and sealed to minimize any remaining air space. The chain of custody during sample collection, transport, and storage should be recorded.

Upon collection, warm (>7 °C) samples of effluent must be cooled to 1 to 7 °C with regular ice (not dry ice) or frozen gel packs. As necessary, ample quantities of regular ice, gel packs, or other means of refrigeration must be included in the transport container to maintain sample temperature within 1 to 7 °C during transit. Samples must be kept from freezing during transport or storage. Temperature can be monitored during transit using max/min thermometers.

Testing of effluent samples should start as soon as possible after collection, and must begin no later than three days after sampling.

Upon arrival at the laboratory, the temperature of the sample must be measured and recorded and must be between 1 to 7 °C upon arrival. The salinity of the sample must also be measured and recorded at this time. Refractometry or conductivity must be used to measure salinity for the purposes of this add-on procedure; guidance for measuring salinity using these techniques can be found in Section 4.2 of ECCC (2017, 2019).^{Footnote 9} Measurements of pH and dissolved oxygen should be recorded on receipt of the effluent sample, before salinity adjustment to 30 ± 2‰ (see Section 4.2.2).^{Footnote 10} After any necessary salinity adjustment of the sample, an aliquot of effluent required at that time may be adjusted immediately or overnight to the test temperature, and used in the test (see Section 4.2.2). Any remaining portion(s) of sample to be stored for subsequent use must be held in darkness in sealed containers, without air headspace, at 4 ± 2 °C.

Table 2: Checklist of required and recommended test conditions and procedures for toxicity tests using Ceramium tenuicorne for the purposes of Canadian-regulated sublethal toxicity testing

Test type

• - 7-day growth inhibition test

Test vessel

• - sterile polystyrene or glass Petri dish with lid
• - 50 × 10 mm, 50 × 9 mm, or 50 × 15 mm

Test volume

• - 10 mL/replicate

Control/dilution water

• - uncontaminated natural seawater (filtered through a paper filter [30 µm]) or artificial seawater; sterilized by autoclave or sterile filtration [0.2 µm] before use; pH-adjusted to 8.0 ± 0.2 using 1 mol/L HCl or 1 mol/L NaOH, both before and after sterilization, if necessary
• - nutrient solutions added to sterilized natural seawater or artificial seawater according to Column B or C, respectively, of Table 3 in ISO (2010)
• - 30 ± 2‰ salinity

Donor vessel preparation to obtain organisms for testing

• - algae tips added to donor vessels with fresh culture media 3 to 4 days before testing begins
• - 20 to 30 algae tips, each approximately 10 to 15 mm long, per donor vessel
• - donor vessels are maintained under normal culturing conditions
• - preparation of 3 to 4 (recommend ≥5) donor vessels should provide sufficient starting material for a test (minimum 64–72 algae tips needed)

Test organism

• - Ceramium tenuicorne marine clone female gametophytes from a culture that has been acclimated at test salinity and temperature for ≥ 2 weeks
• - algae tips cut from organisms in donor vessels
• - each algae tip must be 0.6 to 1.8 mm long and should be 0.6 to 1.2 mm long, measured from the base forking to the most distant tip of the organism; recommend two levels of forking (i.e., tip with two “claws”), but one level of forking is acceptable (i.e., tip lacking “claws”)
• - algae tips must appear healthy (i.e., red-brown, not discoloured); damaged algae tips are discarded; recommend using algae tips of uniform length
• - 2 test organisms/replicate

Number of concentrations

• - ≥ 7, plus dilution-water control
• - additional control treatment (i.e., “salt control” or “hypersaline brine control”) must be prepared if the salinity of the effluent sample is adjusted (see EC, 2001)

Number of replicates

• - ≥ 4 replicates/treatment, including control(s)

Randomization

• - treatments positioned randomly within the test facility
• - recommend changing position of test vessels within the test facility regularly during the test (i.e., once daily, randomly)

Sample requirement

• - 1-L sample of effluent

Sample transport and storage

• - if warm (>7 °C), cool to 1 to 7 °C with regular ice (not dry ice) or frozen gel packs upon collection; sample must not freeze during transit or storage; temperature on arrival must be 1 to 7 °C ; store in the dark at 4 ± 2 °C; use in testing should begin as soon as possible after collection and must begin no later than 3 days after sampling

Salinity

• - if necessary, salinity of effluent sample must be adjusted to 30 ± 2‰ as soon as possible after the sample is received according to guidance in Environment Canada (2001); salt control or hypersaline brine control prepared if salinity of effluent sample is adjusted
• - all test solutions within the range of 28 to 32‰ salinity

Temperature

• - 22 ± 2 °C

Filtration

• - effluent samples are not normally filtered

pH

• - within the range of 7.0 to 9.0; adjusted, if necessary, to within this range using 1 mol/L HCl or 1 mol/L NaOH

Nutrient spiking of effluent

• - nutrients (i.e., nitrogen, phosphorus, iron, and carbon solutions) added to undiluted effluent sample according to Column C of Table 3 in ISO (2010)

Aeration

• - nutrient-spiked effluent samples must be gently pre-aerated for 20 minutes at a minimal rate (i.e., ≤100 bubbles/min) before test initiation
• - test solutions are not aerated during the test

Lighting

• - “daylight” or “warm white” fluorescent or equivalent light
• - intensity of 70 ± 7 μmol m^-2 s^-1
• - photoperiod of 14 h light : 10 h dark

Observations and measurements

• - initial lengths (mm) of a representative sample of algae (≥20 algae tips) at the start of the test (Day 0), measured from the first forking point to the most distant tip of each organism
• - final length (mm) of each algae tip at test end (Day 7), measured from the first forking point to the most distant tip of each organism
• - appearance of algae at start and end of test (Day 0 and Day 7)
• - temperature and salinity measured upon receipt of effluent sample
• - temperature measured daily during test
• - pH, salinity, and dissolved oxygen measured at start and end of test in representative concentrations (minimum control(s), low, medium, and high)
• - light fluence rate (intensity) measured at several locations in the test area at minimum of once during the test (recommend daily)

Endpoints

• - mean increase in length per day (µ) (± SD) at test end for each test concentration and each control treatment
• - IC25 (with its 95% confidence limits) for growth inhibition compared to dilution-water control, based on mean increase in length per day (µ)
• - statistically significant stimulation compared to dilution-water control is reported

Tests with a reference toxicant

• - 7-day test started within 14 days of test period; performed under the same conditions as the definitive test; recommend zinc sulphate (ZnSO₄·7H₂O)

Test validity

• - valid if: (i) increase in length of controls at the end of the test has increased by a factor > 3 compared to the starting length, and (ii) control pH is in the range of 6.5 to 9.5 (inclusive) at test end
• - if more than one control treatment is used, each independently meets all validity criteria

The information in this table is for summary purposes only. Definitive requirements and recommendations of this add-on procedure are contained in the main body of this document.

4.2 Preparing test solutions

All test vessels, measurement devices, stirring equipment, and apparatus for transferring organisms must be non-toxic and thoroughly cleaned and rinsed in accordance with standard operating procedures. Control/dilution water should be used as the final rinse water for items that are to be used immediately in setting up the test; distilled or deionized water should be used as the final rinse for items that are to be stored after allowing them to dry (see EC [2007a] for recommended glassware cleaning procedures).

4.2.1 Control/dilution water

Control/dilution water must be prepared using either uncontaminated natural seawater or artificial seawater. The salinity of natural or artificial seawater used as control/dilution water must be 30 ± 2‰. Uncontaminated natural seawater must be filtered, pH-adjusted if necessary, and sterilized before use (see Section 3.4 of this add-on procedure and Section 4.2.3 of ISO, 2010). Instructions for preparing artificial seawater are found in Section 5.0 of Environment Canada (2001); artificial seawater must be pH-adjusted, if necessary, and sterilized before use (see Section 3.4 of this add-on procedure and Section 4.2.2 of ISO, 2010 for guidance). Nutrient solutions must be added to sterilized natural or artificial seawater according to the concentrations in Column B or C, respectively, of Table 3 in ISO (2010). A given batch of dilution water (natural or artificial) should not be used for more than 14 days following preparation, during which time its container should be kept covered and the contents protected from light (EC, 2001). During prolonged storage (>1 day), natural or artificial seawater prepared for use as dilution water should be refrigerated (4 ± 2 °C) to minimize microbial growth (EC, 2001). The temperature of control/dilution water must be adjusted as necessary to 22 ± 2 °C before its use in preparation of test solutions.

4.2.2 Effluent sample

The salinity of the effluent sample must be adjusted, if necessary, to within the range required for testing (i.e., 30 ± 2‰) following the guidance provided in Environment Canada (2001) as soon as possible following the receipt of the effluent in the laboratory and before test concentrations are prepared. Salinity adjustment with dry salts is recommended, since the 100% effluent can be tested (undiluted) with this option; in addition, most validation work for this add-on procedure was performed using this type of salinity adjustment (see footnote 14). The sample must not be warmed to the test temperature before salinity adjustment occurs (EC, 2001). After any salinity adjustment, the sample temperature must be checked and adjusted, if necessary, to 22 ± 2 °C before starting the test. Sample or test solutions may be adjusted to the test temperature by heating or chilling in a water bath, but must not be heated by immersion heaters or microwaves. Filtration of effluent samples is not normally required. If filtration is considered necessary to mitigate any potential interferences with the test, it must be justified and included as a test deviation. If filtration is used, parallel testing must be performed, so that both filtered and unfiltered samples are tested simultaneously.^{Footnote 11}

After any temperature adjustment, the pH of the effluent sample must be checked; if necessary, the pH must be adjusted to within the range of 7.0 to 9.0 using 1 mol/L HCl or 1 mol/L NaOH.

After any pH adjustment, nutrients (i.e., nitrogen, phosphorus, iron, and carbon solutions) must be added to the effluent sample according to column C of Table 3 in ISO (2010). The nutrient-spiked effluent sample is regarded as the 100% concentration for this test.^{Footnote 12} Prior to preparing test solutions, the nutrient-spiked effluent sample is then gently pre-aerated for 20 minutes at a rate not exceeding 100 bubbles/minute.^{Footnote 13} Oil-free compressed air should be dispensed through airline tubing and a disposable plastic or glass tube (e.g., capillary tubing or a pipette with an Eppendorf tip) with a small aperture (e.g., 0.5 mm inside diameter).

4.2.3 Test concentrations

A minimum of seven test concentrations, in addition to a dilution-water control, must be included in the test. An appropriate geometric dilution series should be used; refer to Section 7.3 of ISO (2010) for guidance on choosing a concentration series. An additional control treatment must be prepared if the salinity of the effluent is adjusted (i.e., “salt control” or “hypersaline brine control”^{Footnote 14}), according to guidance in Environment Canada (2001). Each treatment, including the control(s), must include a minimum of four replicates.

Test solutions must be prepared on the day they are to be used;^{Footnote 15} refer to Section 7.2.3.2 of ISO (2010). Each test solution must be mixed well. For a given test, the same sterilized, nutrient-spiked control/dilution water (test media) must be used for preparing dilutions and the dilution-water control. All test solutions must be within the range of 28 to 32‰ throughout the test (EC, 2001), as measured in representative concentrations (see Section 4.5).

4.3 Preparing organisms for testing

Three to four days before a test is to begin, Ceramium tenuicorne female gametophytes are prepared for use in a test by transferring 20 to 30 algae tips that are each approximately 10 to 15 mm in total length to a clean culture vessel containing 25 mL of fresh, sterile culture media (see Sections 3.3 and 3.4 of this add-on procedure). These are referred to as “donor” vessels. The ISO (2010) standard recommends preparing 3 to 4 donor vessels for each test to obtain a sufficient number of organisms of uniform size for beginning a test (Section 7.1 of ISO, 2010); however, Canadian research experience suggests that ≥5 donor vessels should be prepared for each test to increase the likelihood of obtaining algae tips of uniform size (AquaTox, 2022, 2023). Donor vessels are maintained under normal culturing conditions and algae must appear healthy before being used for setting up a test (see Section 3 and Appendix D of this add-on procedure).

On the day that the test is to begin (i.e., Day 0), a sufficient number of algae tips must be prepared from the donor vessels. The length of each algae tip used to start a test must be 0.6–1.8 mm and should be 0.6–1.2 mm, as measured from the base (first) forking to the most distant tip of the algae (Figure 3).^{Footnote 16} A minimum of 64–72 algae pieces are needed for the multi-concentration test described in this add-on procedure with ≥7 concentrations plus control(s); however, preparing a surplus is recommended to obtain a sufficient number of test organisms of similar size and appearance for use in the test. Section 7.1 of ISO (2010) states that algae tips of the C. tenuicorne marine clone must be cut with two levels of forking, as shown in Figure 1a of the standard. However, in a Canadian laboratory, it was observed that most C. tenuicorne marine clone tips did not have a second level of forking (i.e., “claws”) at the time when the test was initiated (Figure 4; AquaTox, 2022).^{Footnote 17}  Based on Canadian research, sufficient healthy growth (including claw development) that meets validity criteria can be achieved in control organisms during the 7-day test regardless of whether the starting algae tips have zero, one, or two claws (AquaTox, 2022). Therefore, although starting with two claws per algae tip is recommended, it is not a requirement of this add-on procedure for each algae tip to have two claws at test initiation.

Algae pieces can be cut to the appropriate size using a scalpel (ISO, 2010) or miniature scissors (e.g., Exelta Corp® micro self-opening scissors, catalogue no. 63042-032, VWR) and tweezers (e.g., Exelta Corp® curved very fine tip tweezers, catalogue no. 63042-976, VWR) (AquaTox, 2022). The algae pieces should be cut while under a stereomicroscope (ISO, 2010). The cut algae tips are collected in a Petri dish containing sterile culture media or dilution water with salinity of 30 ± 2‰; the most deviating algae tips should be discarded (ISO, 2010). Discoloured (i.e., yellowed or translucent; see Appendix D) or damaged algae tips must not be used to initiate a test.^{Footnote 18} 

The initial lengths of a representative sample of algae tips (≥20) must be measured and recorded before the test is initiated (see Section 7.1 of ISO [2010] and Section 4.4 of this add-on procedure). The initial lengths should be measured under a stereomicroscope, with graph paper under the collecting Petri dish (ISO, 2010). The use of a National Institute of Standards and Technology (NIST) traceable stage micrometer with divisions (e.g., 0.1 mm) may be useful for measuring algae more precisely. Alternatively, an eyepiece can be affixed to the microscope to determine length measurements in micrometre units; these lengths are then converted to millimetre units (AquaTox, 2022). The algae tips that are not part of the representative sample may be cut in the same manner while viewing with a magnifying glass instead of a stereomicroscope, to increase the efficiency of test set up (AquaTox, 2022).

4.4 Test conditions

The duration of the Ceramium tenuicorne growth inhibition test is 7 days. It is a static test; renewal of test solutions is not permitted. The recommended test vessels are sterile, uncontaminated plastic or glass Petri dishes with lids, 50 mm × 10 mm in size (ISO, 2010); 50 × 9 mm or 50 × 15 mm are also acceptable sizes.^{Footnote 19} An identical measured volume of 10 mL of solution must be added to each replicate test vessel. A minimum of four replicates per treatment, including the control(s), are required. A minimum of seven test concentrations, in addition to the control(s), are required.

Tests are initiated by transferring two tips of Ceramium tenuicorne marine clone female gametophytes to each prepared test vessel. Each algae tip must be 0.6–1.8 mm and should be 0.6–1.2 mm in length from the base (first) forking point to the most distant tip, and all algae tips used in a test should be uniform in size and appearance (see Section 4.3). The transfer of algae tips must follow an informal random procedure (for example, see Section 7.4 of ISO, 2010) and care must be taken not to contaminate the algae designated for use in the test while transferring the organisms to their individual test vessels. Each test vessel must be clearly coded or labelled to identify the test material or substance and concentration being tested, and the date and time of starting. Algae tips must be completely submerged in test solution at test initiation and must remain submerged throughout the test. Treatments must be positioned randomly within the test facility, and the position of test vessels within the test facility should be changed regularly during the test (i.e., once daily, randomly).

The test must be conducted at a temperature of 22 ± 2 °C. The following lighting conditions are required during the test: “daylight” or “warm white” fluorescent or equivalent light; intensity of 70 ± 7 µmol m^-2 s^-1 (see footnote 4) at points approximately the same distance from the light source as the test organisms; and photoperiod of 14 h light : 10 h dark.^{Footnote 20} Test solutions must not be aerated during the test. The test is ended seven days after initiation.

4.5 Observations and measurements

Colour, turbidity, odour, and floating or settling solids in effluent samples should be observed and recorded at the start of the test, as well as before and after filtering if applicable. The general appearance of samples and any changes that occur during the preparation of the test solutions should be recorded (e.g., precipitation, flocculation, change in colour or odour, release of volatiles), as well as any changes in the appearance of test solutions observed during the test period.

The initial lengths (mm) of a representative sample of algae (≥20 organisms) must be measured and recorded at the start of the test, as described in Section 4.2 of this add-on procedure, and used to calculate the mean initial length. The final length (mm) of each test organism at the end of the test (Day 7) must be measured and recorded for each replicate of each test concentration and each control treatment. Algae length must be measured from the first forking point to the most distant tip. The final lengths can be measured using a stereomicroscope with graph paper under the Petri dish (ISO, 2010). Measurement of final length using computer image analysis (e.g., ImageJ) is an acceptable alternative technique; however, before making such measurements, the alternative technique must be validated by conducting side-by-side tests comparing length measurements using computer image analysis to those made using a stereomicroscope, and demonstrating that the alternative technique provides a consistent and reliable measurement of algal length.

The algae in each test vessel must be observed at the start and end of the test (Day 0 and Day 7), as a minimum. Observations of test organism appearance such as discoloration (see Appendix D), fragmentation, changes to appearance of cortical bands or axial cells, lack of “claw” development, stunted growth, or disintegrating tips (Figure 5), and other abnormalities should be made and recorded for each test vessel.

Temperature must be monitored throughout the test. As a minimum, temperature must be measured and recorded daily throughout the test. Extra test vessels may be prepared for the purpose of measuring water temperature during the test. If temperature records are based on measurements other than in the test vessels (e.g., in an incubator or temperature-controlled room within the vicinity of the test vessels), the relationship between these readings and temperature within the vessels must be established. Continuous recordings or daily measurements of the maximum and minimum temperatures are acceptable options.

Salinity, temperature, pH, and dissolved oxygen (in both mg/L and percent saturation) must be measured and recorded at the start and end of the test in at least the low, medium, and high concentrations, and the control(s). Measurements should be made on freshly prepared test solutions at test start and on samples of old (used) test solutions pooled from replicates of each treatment at test end, after biological observations are complete.

Light fluence rate (intensity) must be measured at least once during the test period at points approximately the same distance from the light source as the test organisms and at several locations in the test area. However, daily measurements of light intensity are recommended (AquaTox, 2022).

4.6 Test endpoints and calculations

The biological endpoint of this test is based on the growth inhibition of algae exposed to effluent. The measurement endpoint is the increase in length of the algae per day (µ), which is a growth rate calculated according to Equation 1.^{Footnote 21} For each test concentration, including the control(s), the mean (± standard deviation [SD]) increase in length of the algae per day (μ) at the end of the test must be calculated (Section 9 of ISO, 2010). The coefficient of variation (CV = 100 × standard deviation/mean) of the increase in length of the algae per day (μ) for each control treatment must be calculated (see Appendix B), and should be ≤ 30% (see footnote 26). Vessels that have algae tips accidentally removed or stuck and dried to their sides during the test should be removed from the test and that replicate should be excluded from endpoint calculations. In keeping with best practices with evaluation of outliers (see Section 4.6), any vessels excluded from endpoint calculations must be noted in the test report.

µ=  (L₇ – L₀)/(t₇ – t₀) [Equation 1]

where:

µ = increase in length per day (i.e., growth rate)
L₀ = average initial length of organism based on the representative sample on day 0 (mm)
L₇ = final length of each organism in each replicate on day 7 (mm)
t₀ = day 0
t₇ = day 7

For any toxicity test that includes a salt control or a hypersaline brine control in addition to a dilution-water control (see Section 4.2.3 of this add-on procedure), and where responses in both control treatments have met the validity criteria (see Section 4.7 of this add-on procedure), the results of the two sets of control solutions must be pooled before calculating any statistical endpoints (i.e., IC25) involving comparisons of the findings for each set of test concentrations versus those for control solutions (EC, 2001). If there is no overlap between the range in growth rates between the two sets of controls, this must be noted on the test report.^{Footnote 22}

For the purposes of Canadian-regulated sublethal toxicity testing, I_µi and all calculations and interpretations outlined in Sections 9.2–9.4 of ISO (2010) are not required. Instead, endpoint calculations based on quantitative test observations of algal growth must be aligned with other ECCC standardized methods.

The required statistical endpoint for growth data is the inhibiting concentration for a 25% effect (IC25)^{Footnote 23} and its 95% confidence limits (i.e., a 25% reduction in increase in length per day (µ)). For derivation of the IC25 and its confidence limits, the quantitative measurement endpoints are used directly. For this test, the input data must be the mean increase in length per day (µ) for each replicate. EC (2005) provides direction and advice for calculating the ICp, including decision flowcharts to guide the selection of appropriate statistical tests. All statistical tests used to derive endpoints require that the concentrations be entered as logarithms.

An initial plot of the raw data for increase in length against the logarithm of concentration is highly recommended, both for a visual representation of the data, and to check for reasonable results by comparison with later statistical computations. Any major disparity between the approximate graphic IC25 and the subsequent computer-derived IC25 must be resolved. The graph would also show whether a logical relationship was obtained between log concentration (or, in certain instances, concentration) and effect, a desirable feature of a valid test (EC, 2005).

Regression analysis is the principal statistical technique and must be used for the calculation of the IC25, provided that the assumptions below are met. A number of models are available to assess growth data (using a quantitative statistical test) via linear or nonlinear regression analysis. Use of regression techniques requires that the data meet assumptions of normality and homoscedasticity. Weighting techniques may be applied to achieve the assumption of homoscedasticity. The data are also assessed for outliers using one of the recommended techniques (see Section 10.2 of EC, 2005). Any statistical analyses conducted without outliers should also be conducted with the outliers. Any outliers and the justification for their removal must be reported. Finally, the model with the best fit^{Footnote 24} must be chosen as the most appropriate for generation of the IC25 and associated 95% confidence limits. Endpoints generated by regression analysis must be bracketed by test concentrations; extrapolation of endpoints beyond the highest test concentration is not an acceptable practice.

The ability to mathematically describe hormesis (i.e., a stimulatory or “better than control” response occurring only at low exposure concentrations) in the dose-response curve has been incorporated into regression models for quantitative data (see Section 10.3 of EC, 2005). Data exhibiting hormesis can be entered directly, as the model can accommodate and incorporate all data points; there is no trimming of data points that show a hormetic response.

In the event that the data do not lend themselves to regression analysis (i.e., assumptions of normality and homoscedasticity cannot be met), linear interpolation (e.g., ICPIN; see Section 6.4.3 of EC, 2005) can be used to derive an IC25.

A stimulatory effect (increased response at all concentrations or at high concentrations) must be reported for all concentrations in which significant stimulation was observed. If a stimulatory effect was observed, statistical comparison with control(s) is performed using ANOVA analysis, followed by appropriate pairwise comparisons with control(s) (see Sections 3.3 and 7.5 of EC, 2005).^{Footnote 25} This analysis will identify which concentrations show a stimulatory effect that is significantly different from control(s). The percent stimulation for these concentrations must be reported as a test endpoint, using the following calculation (EC, 2007a):

S(%) = (T – C)/C × 100 [Equation 2]

where:

S(%) = percent stimulation
T = mean increase in length per day at test end (µ) in the test concentration (mm)
C = mean increase in length per day at test end (µ) in the control(s) (mm)

4.7 Test validity criteria

For the purposes of Canadian regulated sublethal toxicity testing, the following two criteria^{Footnote 26} must be achieved in each set of control(s) at the end of a test for a test to be valid:

i)  the mean increase in length in 7 days in the control must be >3 times greater than the starting length (Equation 3; see Appendix A for sample calculation);

Increasing factor = (L_7,C – L_0,C)/L_0,C [Equation 3]

where:

Increasing factor = mean fold-increase in length of control organisms after 7 days
L_0,C = mean initial length of control organisms based on the representative sample on Day 0 (mm)
L_7,C = mean final length of control organisms on Day 7 (mm)

ii) the final pH of the sample pooled from the control replicates must be in the range of 6.5 to 9.5 (inclusive).

4.8 Tests with a reference toxicant

A reference toxicant must be used to assess the relative sensitivity of the C. tenuicorne culture used in the toxicity test, and the precision and reliability of data produced by the laboratory for that reference toxicant under standardized test conditions, as well as the technical proficiency of the laboratory staff conducting the test (EC, 1990).

The selected reference chemical(s) must be tested in a multi-concentration test started within 14 days before or after the date that the toxicity test is initiated using the laboratory’s established cultures, and upon acclimation of a new batch of C. tenuicorne. The procedures and conditions to be followed are identical to those in Section 4 of this document and as described in EC (1990), except that aliquots of a reference chemical are added to dilution water and tested instead of an effluent. The control/dilution water (test media) used routinely in effluent tests must also be used for the reference toxicity test (see Section 4.2).

Reagent-grade zinc sulphate (ZnSO₄·7H₂O) is recommended for use as the reference toxicant for this test based on Canadian laboratory experience (ISO, 2010; AquaTox, 2022, 2023; Nautilus Environmental, 2024; see Appendix C). Stock solutions of zinc sulphate should be made up on the day of use. Reagent-grade 3,5-dichlorophenol is also a suitable reference toxicant (ISO, 2010). The 7-day IC25 should be calculated for the reference toxicant used. The concentration of zinc should be expressed as µg Zn⁺⁺/L. The concentration of 3,5-dichlorophenol should be expressed as mg 3,5-dichlorophenol/L. The results of several reference toxicity tests performed at a Canadian laboratory using zinc sulphate are provided in Appendix C for reference.

Concentrations of reference toxicant in all stock solutions should be measured chemically using appropriate methods (APHA et al., 2023). Upon preparation of the test solutions, aliquots should be taken from at least the control, low, middle, and high concentrations, and analyzed directly or stored for future analysis should the IC25 be atypical (i.e., outside warning limits). Zinc solutions should be preserved (APHA et al., 2023) before storage. If stored, sample aliquots must be held in the dark at 4 ± 2°C. Stored aliquots requiring chemical measurement should be analyzed promptly upon completion of the toxicity test. It is desirable, but not required, to measure concentrations in the same solutions at the end of the test. Calculations of IC25 should be based on measured concentrations if they are appreciably (i.e., ≥ 20%) different from nominal ones and if the accuracy of the chemical analyses is satisfactory.

Once sufficient data (e.g., minimum of five data points) are available (EC, 1990, 2005), a warning chart that plots values for IC25 must be prepared and continually updated, with each new reference toxicity test. The warning chart should plot logarithm of concentration on the vertical axis against date of the test or test number on the horizontal axis. Each new IC25 for the reference toxicant should be compared with the previously established warning limits; the IC25 is acceptable if it falls within the warning limits (± 2 SD). All calculations of mean and standard deviation must be made on the basis of log(IC25). This represents continued adherence to the assumption by which each IC25 was estimated based on the logarithm of concentrations. The mean of log(IC25), together with its upper and lower warning limits (± 2 SD), as calculated using the available values of log(IC25), are recalculated with each successive IC25 (EC, 1990, 2005). If the test is run frequently, the most recent 20 reference toxicant points may be used to calculate means and warning limits.

The warning chart can be constructed by simply plotting mean and ± 2 SD as the logarithms or, if desired, by converting them to arithmetic values and plotting IC25 and ± 2 SD on a logarithmic scale of concentration. Different approaches to creating a warning chart (e.g., Levey-Jennings, moving average) are acceptable. Warning charts can be used to detect trends over time. Examples of trends that might be observed include an increasing or decreasing trend, several successive points on one side of the mean, changes that are observed at different times of the year, and successive IC25 values outside the ± 2 SD warning limits.

If a particular IC25 falls outside the warning limits, the sensitivity of the C. tenuicorne culture and the performance and precision of the test are suspect. Since this might occur 5% of the time due to chance alone, an outlying value does not necessarily mean that the sensitivity of the C. tenuicorne culture or the precision of the toxicity data produced by the laboratory is in question. Rather, it provides a warning that this might be the case. A thorough check of all culture and test conditions, as well as technical proficiency, is required at this time.

Test results that usually fall within warning limits do not necessarily indicate that a laboratory is generating consistent results. A laboratory that produced extremely variable data for a reference toxicant would have wide warning limits; a new datum point could be within the warning limits but still represent an undesirable variation in results obtained in the test. For guidance on reasonable variation among reference toxicant data (i.e., warning limits for a warning chart), refer to Section 2.8.1 and Appendix F in EC (2005).

If an IC25 fell outside the control limits (mean ± 3 SD), it would be highly probable that the test was unacceptable and should be repeated, with all aspects of the test being carefully scrutinized. If endpoints fell between the control and warning limits more than 5% of the time, a deterioration in precision would be indicated, and again the most recent test should be repeated with careful scrutiny of procedures, conditions, and calculations.

5.0 Reporting requirements

Test reports must include all of the following requirements, which supersede the reporting requirements listed in ISO (2010).

Each test report must indicate if there has been any deviation from any of the requirements delineated in the ECCC add-on procedure and the referenced sections of ISO (2010), and if so, provide details as to the deviation. The reader must be able to establish from the test report whether the conditions and procedures preceding and during the test rendered the results valid and acceptable for the use intended.

The following items must be included in the test report.

5.1 Effluent sample

• description of effluent type (e.g., process effluent, final effluent, etc.), if and as provided to the laboratory personnel;
• information on labelling or coding of sample;
• date and time of sample collection; date and time sample was received at test facility;
• measurement of temperature and salinity of sample upon receipt at test facility; and
• measurements of temperature and salinity of sample, just before its preparation and use in toxicity test.

5.2 Test organisms

• species, clone, sex, and source of culture; origin and strain number of culture, if known;
• indication as to whether test culture is axenic;
• size and section of the organisms used to start the test;
• growth medium used for culturing; and
• any unusual appearance or treatment of the culture before its use in the test.

5.3 Test facilities and apparatus

• name and address of test laboratory;
• name of person(s) performing the test; and
• brief description of test vessels (size, type of material, sterility specification).

5.4 Control/dilution water

• type(s) and source(s) of water used as control and dilution water; and
• type and quantity of any chemical(s) added to control or dilution water.

5.5 Test method

• citation of the biological test methods used (i.e., as per ISO 10710:2010 “Water Quality – Growth inhibition test with the marine and brackish water macroalga Ceramium tenuicorne” with ECCC add-on procedure)
• brief description of procedure(s) if sample received filtration and/or pH adjustment;
• brief description of procedure(s), products used, and duration of aging for any salinity adjustments of sample and control/dilution water, if applicable; statement that the Environment Canada guidance document on salinity adjustment has been followed;
• brief description of frequency and type of observations and measurements made during test;
• name and citation of program(s) and methods used for computer image analysis, if applicable; and
• name and citation of program(s) and methods used for calculating statistical endpoints.

5.6 Test conditions and procedures

• test type (static 7-day test);
• design and description if any deviation from or exclusion of any of the procedures and conditions specified in this ECCC add-on procedure and referenced sections of ISO (2010);
• individual lengths of at least 20 representative organisms at the start of the test (Day 0) and their calculated mean initial length;
• number and concentration of test solutions, including control(s);
• number of replicates per treatment, including control(s);
• volume of solution in each test vessel;
• number of organisms in each test vessel;
• brief statement (including procedure, rate, and duration) of any pre-aeration of sample or test solutions before starting the test;
• type and quantity of chemicals added to test sample before starting the test (i.e., nutrient spiking);
• all required measurements (see Section 4.3) of temperature, salinity, pH, and dissolved oxygen in test solutions including control(s), and measurements of light fluence rate made during the test;
• date and time when test was started and ended; and
• brief statement indicating whether the reference toxicity test was performed under the same experimental conditions as those used with the test sample(s), and description of any deviation(s) from or exclusion(s) of any of the procedures and conditions specified for the reference toxicity test in this add-on procedure.

5.7 Test results

• any observations of organism appearance in each test vessel at the start and end of the test;
• final length of each organism in each test vessel at the end of the test (Day 7);
• mean (± SD) increase in algal length per day (growth rate, µ) for each concentration, including control(s), at the end of the test;
• any ICp and its 95% confidence limits for percent inhibition of growth rate and indication of quantitative method used; details regarding any transformation of data that was required;
• any outliers or vessels excluded from endpoint calculation and justification for their removal;
• mean increasing factor at the end of the test for each control treatment;
• any findings of significant growth stimulation, expressed as % stimulation, at any concentration(s);
• ICp and 95% confidence limits for any toxicity tests with the reference toxicant(s) started within 14 days of the test, together with the geometric mean value (± 2 SD) for the same reference toxicant(s) as derived at the test facility in previous tests; and
• anything unusual about the test, any problems encountered, and any remedial measures taken.

References

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Appendix A: Sample calculation for mean increasing factor in control

Table A.1 provides a sample calculation for the increasing factor in the dilution-water control treatment at test end, using data from a Canadian laboratory following this add-on procedure (AquaTox, 2023). Equation 3 (Section 4.7) is used to calculate the mean increasing factor. In this example, the mean increasing factor for algal length in the dilution-water control treatment after 7 days was 24.76. This value is >3 and therefore meets this validity criterion (see Section 4.7 of this add-on procedure and Section 8 of ISO [2010]).

Appendix B: Sample calculations for control growth rate and coefficient of variation

Sample calculations for mean increase in length per day (µ; see Equation 1 in Section 4.6) and its coefficient of variation (CV = 100 × standard deviation/mean) in the dilution-water control treatment, using data from a Canadian laboratory following this add-on procedure (AquaTox, 2023). The CV of the control growth rates is ≤ 30%, which meets the recommendation in Section 4.6 of this add-on procedure.

Appendix C: Zinc sulphate reference toxicity test results from a Canadian laboratory

^* The lab also calculated the IC50 for tests 1–5, and the endpoints are as follows: 37.0, 85.3, 40.9, 65.5, and 83.6 μg Zn⁺⁺/L, which is equivalent to 0.163, 0.375, 0.180, 0.288, and 0.368 mg ZnSO₄·7H₂O/L, for tests 1, 2, 3, 4, and 5, respectively.

Appendix D: Culture health, sex identification, and discolouration

Dixon (1960) and Cho et al. (2001) describe the structure of the genus Ceramium. The culture of Ceramium tenuicorne female gametophytes used for method validation and deposited to the NCMA culture bank were sourced from the University of Oslo, where it had been maintained in culture since the 1970s (Rueness, 1973); the species and sex of the culture were confirmed by the late Dr. Stein Fredriksen. In order to identify the sex of Ceramium for cultures from a different source, it is necessary to see the sexual structures. On males, spermatia are released from spermatangia, which can be observed as round, colourless structures covering each cortical band; males may also have unicellular, long, thin hairs (Figure D.1). Females develop structures that may be harder to identify: i) procarps are a group of four cells and an elongated structure called a trichogyne, which is the receptor for a male spermatium; ii) after fertilization, the female will develop cystocarp(s) (Figure D.2); cystocarps produce carpospores, which develop into tetrasporophytes (Dixon, 1960; Eklund, 1998, see Figure 1; Cho et al., 2001; S. Fredriksen, University of Oslo, personal communication, 2021). Without sexual structures present it is impossible to separate the sexes (haploid gametophytes) from each other, and they will look similar to a diploid tetrasporophyte with no tetrasporangia present (S. Fredriksen, University of Oslo, personal communication, 2021).

The Norwegian Culture Collection of Algae (NORCCA) was originally used as a source of Ceramium tenuicorne culture for Canadian research; however, NORCCA was not able to confirm the sex of the algae at the time. Regardless of growth media, salinity, or light intensity used, algae sourced from NORCCA grew slowly and would clump within test vessels (AquaTox, 2022). Based on photographic comparison of the male (Figure D.1) and female (Figure D.2) gametophytes cultured at the University of Oslo, it was suspected that male gametophytes had been obtained from NORCCA unintentionally (Figure D.3) (AquaTox, 2022). A confirmed culture of female gametophytes was subsequently sourced from the University of Oslo (S. Fredriksen, University of Oslo, Norway). This culture grew at a faster rate in comparison to the algae from NORCCA, without clumping or developing hairs (Figure D.4). Figure D.5 depicts healthy growth of C. tenuicorne culture from the University of Oslo after two weeks of culturing (see Figure 1 for original sizes and structures).

The Canadian lab experienced one incidence of contamination in their C. tenuicorne culture throughout method validation (Nautilus Environmental, 2024). The contamination was present in 93% of culture vessels. It visually appeared to be small, dense clusters of non-mobile cells adhered to the bottom of the vessel (Figure D.6), but the identity and source of the contaminant were unknown. The contamination was not observed in any of the tests completed during method validation, and further tests were not initiated until the contamination was resolved through supplementation with organisms from the mass culture (Nautilus Environmental, 2024).

Discolouration, including yellowing (chlorosis) and translucent algae, has been observed in C. tenuicorne cultures and tests in a Canadian laboratory (M. Gallant, Nautilus Environmental, personal communication, 2023; Nautilus Environmental, 2024), and at the NCMA culture bank (K. Dykens, NCMA, personal communication, 2023); refer to footnote 18 for details. Figure D.7 depicts an example of translucent algae observed at Nautilus Environmental (2024); the algae tip may have been damaged at test initiation, as it did not grow during the 7-day test period. Figure D.8 depicts examples of the yellowed algae observed at the NCMA, which were healthy and red at the tips but yellow toward the base.