Ecotoxicological Assessment of Antifouling Biocides and Nonbiocidal Antifouling Paints

Appendix 2: Examination of the mineralization of DCOI and zinc pyrithione in marine sediments

1. Introduction
2. Materials and methods
3. Results

1. Introduction

The mineralization of 4,5-dichloro-2-n-octyl-4-isothiazolin-3-on (DCOI) and zinc pyrithione was examined in laboratory tests with marine coastal sediments. The tests were performed under aerobic and anaerobic conditions using test concentrations of ng/g, which is assumed to result in environmentally realistic transformation kinetics. In the anaerobic experiments, sulfate-reducing conditions were established by adding sulfate. Marine coastal sediments contain considerable amounts of sulfate (Sørensen et al. 1979). Glucose was included in the tests in order to examine the mineralization of a readily biodegradable substance at low concentrations and under the given test conditions.

2. Materials and methods

Sediment and seawater
Sediment samples and their seawater were collected at two localities in the Sound. The two sets of sediment and water samples can be described as follows:

Clayey sediment (LS)

	Location: The Sound, 55° 50'642N - 12° 40'854E; depth (sediment), 22 m; depth (water), 20 m.
	Texture: coarse sand (0.25-2 mm), 0.9%; fine sand (0.063-0.25 mm), 26.4%; silt and clay (<0.063 mm), 65.8%; organic matter, 6.9%.
	Number of bacteria (water): 3.1 × 103 per mL.
	Number of bacteria (sediment): 1.5 × 105 per g.

Sandy sediment (SS)

	Location: The Sound, 55° 50'030N - 12° 37'534E; depth (sediment), 4 m; depth (water), 2 m.
	Texture: coarse sand, 91.5%; fine sand, 7.3%; silt and clay, 1.0%; organic matter, 0.2%.
	Number of bacteria (water): 2.1 × 103 per mL.
	Number of bacteria (sediment): 4.8 × 104 per g.

The number of bacteria in seawater and sediment was determined as the bacterial count by embedding a known amount of the sample in Bacto Marine Agar 2216 (Difco). Sediment (approx. 1.6 g) is mixed with 9 mL fosfate buffer and is then treated as a water sample. The bacterial count is determined as the number of colonies occurring after 72 hours’ incubation at 21°C. Sediment and water samples were stored separately stored in the dark at 4°C until use.

Chemicals
[2,3-¹⁴C]DCOI (Lot Nos. 853.0208 and 853.0209; 15.9 mCi/mmol, 56.44 µCi/mg, radio-chemical purity 98.6%) was supplied by Rohm and Haas Research Laboratories (Spring House, Pennsylvania). [¹⁴C]zinc pyrithione (Lot. No. 3228-143; 157.63 mCi/mmol, 0.50 mCi/mg) and [UL-¹⁴C]D-glucose (Lot 48H9476 Sigma; 212.5 mCi/mmol, 1.18 mCi/mg, dissolved in ethanol:water, 9:1) was supplied by Arch Chemicals (Cheshire, Connecticut). All other chemicals are commercially available and of analytical purity.

Aerobic biodegradation tests
The aerobic biodegradation tests were made with sediment LS as well as sediment SS. Stock solutions of [¹⁴C]DCOI (in methanol), [¹⁴C]zinc pyrithione (in dimethyl sulfoxid) and [¹⁴C]glucose (in deionized water) were added to 300-mL glass flasks with 10 g sediment (wet weight) and 50 mL seawater which had beforehand been aerated with atmospheric air for approx. 20 hours. For [¹⁴C]DCOI, the stock solution was added to the test flasks with 0,5 g dried sediment and the methanol was allowed to evaporate before more sediment and water were added. The other substances were added by mixing the stock solution with the sediment, after which seawater was added. The resulting concentrations of the three model compounds are indicated in Table B2.1. Two glass pipes were placed in the test flasks, one pipe with 1N KOH (3 mL) for absorption of ¹⁴CO₂ and another with ethylene glycol for trapping other gaseous compounds. The flasks were closed with rubber stoppers and aluminium screw caps and placed in the dark at 15°C.

The mineralization of the substances was followed by determining the ¹⁴C activity which was trapped in the glass pipes. Each week, samples were taken for liquid scintillation counting and the test flasks were placed in the dark without stoppers and caps for approx. 10 min in order to exchange the gas phase of the flasks with atmospheric air. Then the contents of the glass pipes were replaced with fresh KOH or ethylene glycol. At the end of the test after 42 days, CO₂ in the water phase was released after acidification of the sediment-water system to pH 1-2 by addition of concentrated sulfuric acid.

Anaerobic biodegradation tests
The anaerobic biodegradation tests were only made with sediment LS. In the same way as in the aerobic tests, the stock solutions of the three model compounds were added to 117-mL serum flasks with 30 g of sediment (wet weight) and 30 mL seawater. The resulting concentrations of the three model compounds are indicated in Table B2.1. Before use, the seawater was pre-treated with an addition of a fosfate buffer (27 mg KH₂PO₄ and 112 mg Na₂HPO₄ × 12H₂O per litre) in order to stabilize pH and a redox indicator (1.0 mg resazurin per litre) in order to control that anaerobic conditions were present throughout the test. Sulfide (0.5 g Na₂S × 9H₂O/ kg) was added to each serum flask as reducing agent while sulfate (Na₂SO₄; 25 mM in the water phase) was added as electron acceptor in order to establish sulfate-reducing conditions, which are characteristic of marine sediments. A glass pipe with 1N KOH (3 mL) was placed in the serum flasks for absorption of ¹⁴CO₂. Throughout all of the above procedure, the serum flasks were carefully aerated with oxygen-free N₂ gas until they were closed with 1-cm butyl rubber stoppers and aluminium screw caps. The test flasks were placed in the dark at 15°C

The mineralization of the substances was followed by determining the ¹⁴C activity (from ¹⁴CO₂) which was trapped in the glass pipe with KOH. Sampling of KOH for liquid scintillation counting was made after 14, 28 and 56 days after which the liquid in the glass pipe was replaced with fresh KOH. As part of the carbon at the mineralization of the model compounds may be transformed into methane, ¹⁴CH₄ was determined by injecting 2-mL gas samples into scintillation vials which were modified so that the caps could hold a septum (Zehnder et al. 1979). Before use, a hole was made in the screw cap of each scintillation vial and a butyl rubber septum inserted. 20 mL of a liquid scintillation cocktail (Insta gel II plus, Packard) was added to the scintillation vials. It turned out that the cocktail was capable of absorbing approx. 2/3 of the methane added in pilot tests. At the end of the test after 56 days, CO₂ in the water phase was released after acidification of the sediment-water system to pH 1-2 by addition of concentrated sulfuric acid.

Table B2.1
Aerobic and anaerobic mineralization. Initial concentrations of model compounds.

Recovery of remaining ¹⁴C
After the termination of the aerobic and anaerobic tests, the ¹⁴C remaining in water sediment was determined by the following procedures. In the test, in which [¹⁴C]DCOI had been added at a concentration of 0.033 µg/g, and in the tests with [¹⁴C]glucose, the remaining radioactivity was determined after liquid scintillation counting of sub-sample of the water phase and incineration of 0.1-g sediment samples in excess of oxygen. In the other tests, the ¹⁴C remaining in the sediment was determined by extraction with 6N HCl followed by 1N NaOH, after which the extracted sediment was incinerated in excess of oxygen (Madsen and Kristensen 1997). This method makes it possible to determine fractions of ¹⁴C, which are bound in the form of hydrolyzable compounds, humic and fulvic acids or to humin/ clay minerals.

Chemical analyses
DCOI and metabolites from the transformation of DCOI were analyzed and characterized by Rohm and Haas (Andrew Jacobson, Rohm and Haas Research Laboratories, Spring House, Pennsylvania). Water samples were treated with solid phase extraction (SPE) by use of acetate:methanol (1:1) before analysis in HPLC. Sediment samples were extracted twice with acetonitril: 0.01 N HCl (4:1) followed by extraction with dichloromethane for analysis in HPLC.

Zinc pyrithione and its metabolites were determined by Arch Chemicals (James C. Ritter, Department of Ecotoxicology, Cheshire, Connecticut). Sediment samples were extracted with acetonitril followed by two extractions with 1,0 N KOH. Then the water samples and the extracts from sediment were derived before analysis in HPLC (Arch Chemicals 1999b).

3. Results

The cumulated development of ¹⁴CO₂ from the mineralization of DCOI and zinc pyrithione is shown in the Figures 3.1-3.3 (Section 3.2 of the main report) and Figures 4.1-4.3 (Section 4.3 of the main report). The total mineralization and distribution of the radioactivity remaining at the end of the tests are shown in Tables B2.2-B2.4. The amount of ¹⁴C in absorbers with ethylene glycol (aerobic tests) constituted <0.1% of the radioactivity added.

Table B2.2    Look here...
Mineralization of DCOI in sediment and seawater under aerobic or anaerobic conditions. Distribution and recovery of ¹⁴C after an incubation of 42 days (aerobic test) or 56 days (anaerobic test).

Table B2.3    Look here...
Mineralization of zinc pyrithione in sediment and seawater under aerobic or anaerobic conditions. Distribution and recovery of ¹⁴C after an incubation of 42 days (aerobic test) or 56 days (anaerobic test).