Ecotoxicological Assessment of Antifouling Biocides and Nonbiocidal Antifouling Paints

Appendix 3: Examination of the effect of degradation and sorption on the aquatic toxicity of DCOI and zinc pyrithione

1. Introduction
2. materials and methods

1. Introduction

The effect of degradation and sorption on the toxicity of 4,5-dichloro-2-n-octyl-4-isothiazolin-3-on (DCOI) and zinc pyrithione was examined in laboratory tests with the marine crustacean Acartia tonsa. As especially zinc pyrithione is degradable by photolysis, the tests were made in the dark as well as at a constant exposure to light.

2. Materials and methods

Sediment and seawater
This test was made with the sandy sediment (SS) and its seawater as described in Appendix 2.

Chemicals
DCOI (Lot No. 14-SS-18F; purity, 99.86%) was supplied by Rohm and Haas Research Laboratories (Spring House, Pennsylvania). Zinc pyrithione (Lot. No. J116659; purity, 98.9%) was supplied by Arch Chemicals (Cheshire, Connecticut).

Bioassays
DCOI and zinc pyrithione were added from stock solutions with methanol (50 µL) and dimethyl sulfoxid (50 µL), respectively, to 55-mL serum flasks with 0.5 g dried sediment. The methanol from the stock solution of DCOI was allowed to evaporate. Seven grammes of sediment (dry weight) was then added together with 35 mL of the respective seawater, which, before addition, had been adjusted to a salinity of 3.2%. The initial concentrations were 0.1 µg/g for DCOI and 0.025 µg/g for zinc pyrithione. As the gas phase of the test system, pure oxygen was added, after which the serum flasks were closed by Teflon-coated rubber septa and aluminium caps. One series of serum flasks were incubated in the dark while a parallel series of flasks were incubated at constant exposure to light. Both series of test flasks were incubated at a temperature of 20 ± 2°C. The light-exposed serum flasks were incubated bottom up and with a distance of 5 cm to fluorescent tubes (Pope FID 18W/33; a total of 14 tubes), which had been mounted with spaces of approx. 1.8 cm in between. The average light intensity at the distance of 5 cm from the light source was measured at 458 ± 32 µmol/m² × s in air, which was extrapolated to 342 µmol/m² × s in water. Measurements performed by VKI in the Sound have shown that the average light intensity in a depth of approx. 1 m (0.6-1.4 m) was 548 µmol/m² × s in 1997 and 420 µmol/m² × s in 1998 (the measurements in 1997 as well as in 1998 were made in the period from 9 May till 30 September by daylight).

Three replicate serum flasks from bioassays were harvested after 0; 1; 2; 4; 7 and 14 days. Before sampling, the flasks were shaken after which they were placed in the dark in order for the sediment to settle. The water phase from each replicate was carefully transferred to centrifugal vials and, after centrifuging (1,500 rpm for 15 min), the supernatant was stored in a deep-freeze until use in the test with A. tonsa.

The acute toxicity to A. tonsa was tested by use of the ISO/FDIS 14669 procedure with the modification that the test was made in the dark in order to prevent transformation by photolysis of zinc pyrithione. Controls in this test included:

	Supernatant (t = 0 d) from test system without biocide
	Supernatant (t = 0 d) from test system without biocide with addition of 50 µL methanol as in the test
	Supernatant (t = 0 d) from test system without biocide with addition of 50 µL dimethyl sulfoxid as in the test
	Supernatant (t = 14 d) from test system without biocide incubated in the dark as in the test
	Supernatant (t = 14 d) from test system without biocide exposed to light as in the test
	Seawater from the Sound (sediment SS; cf. Appendix 2) adjusted to a salinity of 3.2%
	Seawater from the Kattegat adjusted to a salinity of 3.2%

Further information on the toxicity test with A. tonsa is given in the report "Ecotoxicological tests of leachates of antifouling paints" (Bjørnestad et al. 1999).

The effect of dosing with biocides on the number of bacteria in the test system was determined as the bacterial count (cf. Appendix 2). The number of bacteria in an untreated control sample was 9.1 × 10⁵ per mL after 1 day’s incubation while the corresponding numbers were 1.5 × 10⁵ and 1.4 × 10⁵ per mL in samples with dosages of DCOI and zinc pyrithione, respectively.