Distribución espacio temporal de las variables climatológicas

This component of the study focussed on a suite of fouling organisms that we used as indicators of treatment effects. These were Undaria pinnatifida and Ciona intestinalis which are recognised fouling pests, and nine other fouling taxa that are globally widespread (e.g., Furlani, 1996) and represent a range of morphologies (e.g., filamentous, soft-bodied, calcareous) that we regarded as useful surrogates for structurally and functionally similar pests. The latter were solitary tunicates (Cnemidocarpa bicornuata, Corella eumyota), colonial tunicates (Botryllus schlosseri,

Botrylloides leachi), encrusting (Watersipora subtorquata) and erect (Bugula neritina)

bryozoans, tube-dwelling serpulid (Hydroides elegans) and terebellid (Family Terebellidae) polychaetes, and a filamentous green macroalga (Cladophora sp.).

Except for Undaria, we were able to collect the indicator organisms from passive fouling on 1 m lengths of weighted rope suspended for 8 months from marina pontoons. For Undaria it was necessary to create artificial cultures to achieve a sufficient density of plants. The different life-stages of Undaria are relatively amenable to experimental manipulation, therefore working with this species also provided the ability to rapidly evaluate a range of potential methodological approaches prior to treatment of the fouled ropes. Furthermore, Undaria provided the opportunity to assess acetic acid effects on microscopic as well as visible life forms, recognising that while mussel seed-stock

life-stages can survive the process (Forrest and Blakemore, 2003, 2006).

Experiments with Undaria

Initial work with Undaria compared the effects of acetic acid on the survival of gametophytes and plantlets (sporophytes < 50 mm length), and on the viability of reproductive (sporophyll) tissue to account for instances where either mature sporophytes or fragments of sporophyll are transferred with seed-stock (Table 7.1). Acetic acid concentrations mixed in both seawater and fresh water were compared, to determine the most effective diluent for subsequent work. Gametophytes were cultured on sterile 24-well Falcon™ plates, whereas plantlets were cultivated on weathered rope (Forrest and Blakemore, 2006). Sporophylls were collected from a mature Undaria population, with the experimental units comprising a disc (10 mm diameter) of excised tissue held within each of the 24 wells on a Falcon™ plate. These three life-stages were exposed to acetic acid treatments as indicated in Table 7.1, with a post-treatment seawater rinse used to remove any residual chemical.

Gametophyte and plantlet mortality was assessed one week post-treatment using methods described by Forrest and Blakemore (2006). For the sporophyll tissue, the post-treatment procedure involved high humidity (> 95% RH) air exposure for 24 h at 17 o_{C to induce partial dehydration, and then rehydration in filtered (25 µm) UV-}

sterilised seawater at 17 oC. This partly mimicked conditions that Undaria would be exposed to during inter-regional seed-mussel transfer (i.e., high humidity emersion) but, more importantly, was expected to provide optimal conditions for spore release (Saito, 1975). After 1 h of rehydration, the sporophyll disc was removed from each well, and the seawater replaced with a nutrient-enriched growth medium. The effect of the acetic acid treatment on sporophyll tissue viability, defined here as its ability to release competent spores, was assessed as the density of gametophytes attached to the bottom of each well two weeks after treatment. Viability was assigned on a ranked scale (1 – 5) to reflect gametophyte densities in each well as follows: 0 = absent; 1 = 1 – 5; 2 = 6 – 20; 3 = 21 – 50; 4 = 51 – 100; 5 = > 100.

This initial work revealed that sporophyll tissue was more resilient to the effects of acetic acid than were gametophytes or plantlets. Furthermore, while fresh water

Table 7.1 Summary of experiments conducted with Undaria pinnatifida, fouled ropes,

and mussel seed-stock.

Experimental component Acetic acid (%) Exposure time (min) Diluent Experimental temperature (o_C) End-point n Undaria gametophytes 0.1 – 2 1 Seawater & fresh water Ambient Mortality 5 Undaria plantlets 0.1 – 2 1 Seawater & fresh water Ambient Mortality 5 Undaria sporophyll 0.1 – 2 1 Seawater & fresh water

17 o_{C air for 24 h post-} treatment

Viability 1 ₅ Undaria

sporophyll

2 and 4 1, 2, 3, 4 Seawater 17 o_{C air for 24 h post-} treatment

Viability 1 ₄ Fouled ropes

(field) 2

2 and 4 1, 2, 3, 4 Seawater Ambient 6 – 17 o_{C air} for 24 h, seawater 15 – 16 o_{C during} ongrowing Mortality 1–4 Seed mussels (laboratory) 2

4 and 8 2 Seawater 10, 15 and 20 o_{C air for} 24 h

Attachment 4 3 Seed mussels

(field) 2

4 1, 2, 4 Seawater Ambient 11 – 18 o_{C air} for 24 h, seawater 15 – 17 o_{C during}

ongrowing

Survival 3 3

Notes:

1 _{See text for details of sporophyll viability method.}

2 _{Experiments with fouled ropes and mussels included evaluation of ‘rinse’ vs ‘no rinse’ post-treatment.} 3 _{Replicates used in the mussel experiments each comprised 20 mussels for the laboratory work and} approximately 70 mussels for the field trial.

practical convenience of using seawater during routine field operations. Hence, further experiments assessed the effects of seawater dilutions of acetic acid on sporophyll tissue using the method described above, at the concentrations and immersion times indicated in Table 7.1. These experimental conditions were chosen on the basis of pilot work indicating that effective treatments at concentrations of 1% or less would require exposure times > 10 minutes, which would not be practical within the context of many aquaculture operations. The design included a comparison of the effects of a post-

acetic acid on the sporophyll discs prior to their rehydration. The purpose of this work was primarily to identify treatments that were completely lethal to Undaria (i.e., only major changes among the different treatments were of interest), therefore statistical analyses were not conducted.

Experiments with fouled ropes

The 1 m lengths of fouled rope were treated using the same concentrations and exposure times described above for Undaria (Table 7.1), and similarly included a comparison of rinse vs no rinse post-treatment. To understand the limiting processes operating during mussel seed-stock movement, we compared the effects of treatment and transport in isolation, and the combined effects resulting from treatment followed by transport, and vice versa. Transport was simulated by holding treated ropes in plastic bins (covered to simulate high humidity during transport) at ambient temperatures for 24 h (Table 7.1). As a result of the substantial biomass present on the ropes (hundreds of kilograms in total), replicate ropes were included for ‘control’ and ‘transport’ only, with single ropes used for other treatments. While this did not provide a measure of treatment variability, we were nonetheless able to examine consistency in patterns of efficacy with increasing exposure time. After four weeks of on-growing from marina pontoons post-treatment, the wet weight of fouling biomass was measured on each rope, and the fouling indicator species surviving the various treatments were described.