9. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD 9.1 Laboratory experiments VC has been shown to be mutagenic in several in vitro and in vivo test systems derived from organisms belonging to different taxonomic levels. The details referring to bacterial, fungal and mammalian cell lines or to whole organisms like insects or plants are discussed in section 7.6. The carcinogenic effects of VC are addressed in section 7.7. The following chapter focuses on investigations of other signs of toxicity relevant to organisms that may be exposed to VC in the environment. Standard tests on survival and reproduction were not available. Care must be taken when interpreting the toxicity results available as many were obtained from static tests using nominal exposure concentrations. Such tests will have large losses of VC due to volatilization, thus reducing the actual exposure to VC. 9.1.1 Microorganisms 9.1.1.1 Water A consortium of anaerobic microorganisms (species not identified; initially obtained from municipal sludge) was used for testing VC toxicity. Both batch and semi-continuous assays were conducted. VC had an inhibitory effect on the total gas production, beginning at a concentration of 5.4 mg/litre and resulting in an EC50 value of approximately 40 mg/litre, as seen in the batch assay over 3.5 days. In the semi-continuous assay lasting 15 days, the threshold was greater than 64 mg/litre (the highest concentration tested), probably due to volatilization of VC (Stuckey et al., 1980). The growth of five mixed bacterial populations (isolated from natural aquatic systems) was not affected, as compared to controls, in liquid cultures (closed flasks; 21 °C; over 5 weeks) containing up to 900 mg VC/litre (Hill et al., 1976b). The toxicity of waste effluents of a VC production plant to the green alga Chlorella sp. was tested before and after the wastes were 227 EHC 215: Vinyl Chloride ______________________________________________________ treated by neutralization procedures (addition of aqueous solutions of NaOH and catalysts). The crude effluents consisted of VC (15–18%, weight), di-, tri- and pentachloroethanes (45%), dichloroethylene (27%), dichloropropane (6%), ethyl chloride (1%) and unidentified substances (1%). This mixture led to a weak inhibition of algal growth (measured by changes in optical density at 678 nm), with a 72-h EC50 value of 1495 mg/litre (which corresponds to a VC concentration of 224 mg/litre). It should be noted that other compounds present in the effluent may also have contributed to the toxicity. The corresponding EC50 values of the neutralized waste samples (filtrates and extracts of precipitates; composition not analysed) ranged from 4000 to > 100 000 mg/litre (Demkowicz- Dobrzanski et al., 1993). 9.1.1.2 Soil Soil (aquifer) microcosms enriched for methanotrophic activity transformed up to 90% of the 1–17 mg/litre influent VC with no apparent toxic effects (Dolan & McCarty, 1995a,b). However, when both VC and 1,1-dichloroethylene were present, about 75% less transformation of VC and a marked decrease in methane oxidation rate was observed (Dolan & McCarty, 1995a). Toxic effects, measured as decreased methane uptake, were seen during degradation of VC (VC concentrations: up to saturated solutions; solubility: 2.7 mg/ml) by a culture of mixed methanotrophs (seeded with soil from a defunct landfill). A mixture of VC and trichloroethylene (TCE) and a triple mixture of VC, cis-dichloroethylene (c-DCE) and TCE showed cumulative toxicity (Chang & Alvarez-Cohen, 1996). The nitrifying soil bacterium Nitrosomonas europaea had a turnover-dependent loss of (ammonia-dependent) O2 uptake activity after co-metabolic transformation of VC (Rasche et al., 1991). 9.1.2 Aquatic organisms 9.1.2.1 Invertebrates VC reduced the population doubling time of a ciliated protozoon, Tetrahymena pyriformis, population cultured in vitro (Sauvant et al., 1995). The IC50 value was 540 mg/litre (8.6 mmol/litre). 228 Effects on Other Organisms in the Laboratory and Field ______________________________________________________ During a 96-h assay VC had no effect on the survival of the free- living nematode Panagrellus redivivus at concentrations ranging from 10-3 to 10 -8 mol/litre (0.6–62 500 :g/litre), but it reduced the developmental success of this species. The molting rate from the fourth larval stage to adult (determined on progeny of 150 to 300 gravid females per assay; three replications) was significantly decreased relative to controls, and that primarily at VC concentrations of 6.3–6300 :g/litre (Samoiloff et al., 1980). The effects of waste effluents from a VC plant described in section 9.1.1.1 (test with Chlorella sp.) were also tested with the crustacean Daphnia magna (n = 3 × 10 per experiment; age: 6–24 h). The test parameter for the latter was lethality, which was determined by procedures based on ISO 6341-1982 (ISO, 1989). Crude waste produced a 24-h LC50 value of 80.7 mg/litre (related to VC: 12 mg/litre). The neutralized wastes (filtrates and extracts of precipitates) were less toxic, reflected by the higher LC50 values ranging from 445 to > 100 000 mg/litre (Demkowicz-Dobrzanski et al., 1993). 9.1.2.2 Vertebrates The acute toxicity of VC to fish was examined with a few freshwater species; 96-h LC50 values of 1.22 g VC/litre and 1.06 g VC/litre were reported for bluegill (Lepomis macrochirus) and largemouth bass (Micropterus salmoides), respectively, but without giving further details (Hann & Jensen, 1974). Brown et al. (1977) exposed Northern pikes (Esox lucius) to 388 mg VC/litre; 10 days after exposure all test animals (n=15) were dead (versus 1 of 20 in controls during 120 days of observation). However, the test conditions (e.g., handling of controls, water quality) were not sufficiently described in this study. Tests of zebra fish (Brachydanio rerio; n = 10 per group) according to OECD guideline 203 (OECD, 1984), which was adapted to volatile chemicals, resulted in LC50 values (based on mean measured test concentrations) of 240 mg/litre (24 h) or 210 mg/litre (48 h, 72 h and 96 h). The no-observed-effect concentration (NOEC) regarding mortality was 128 mg/litre (Groeneveld et al., 1993). Estimated benchmark values, concentrations believed to be non- hazardous, to freshwater fish derived by several methods ranged from 229 EHC 215: Vinyl Chloride ______________________________________________________ 87.8 to 28 879 :g/litre (Suter, 1996). The Tier II secondary acute value and secondary chronic value were reported to be 1570 :g/litre and 87.8 :g/litre, respectively. The lowest chronic benchmark for fish was estimated to be 28 879 :g/litre, with a corresponding fish EC20 of 14 520 :g/litre. 9.2 Field observations 9.2.1 Aquatic organisms A field study of benthic invertebrates (Dickman et al., 1989; Dickman & Rygiel, 1993) was carried out during 1986–1991 at 15 sites of the Niagara River watershed (Canada) near a PVC plant. The VC discharge in 1986 was estimated to be more than 32 kg (accompanied by unknown quantities of organotin). The density and diversity of several invertebrate groups (Amphipoda, Culicoidae, Chironomidae, Isopoda, Hirudinea, Oligochaeta, Gastropoda, Trichoptera) was found to be low as compared to a reference site. Chironomids (Diptera) turned out to be the best indicators. They were absent at the sampling site closest to the factory’s discharge pipe, and their numbers increased with increasing distances. Those collected nearest to the discharge site primarily belonged to the genus Polypedilum, which is known to be pollution tolerant. Nevertheless, individuals of this genus showed a high proportion of larval mentum (labial plate) deformities (38% versus 1.7–5.7% at reference sites). Altogether, the frequencies of deformities at the discharge site fell significantly (P = 0.05) from 47% measured in 1986–1989 to 25% in 1991, which correlated with the lower levels of VC (figures not given) being released into the river in 1990–1991 (Dickman & Rygiel, 1993). 230 10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT 10.1 Evaluation of human health effects 10.1.1 Hazard identification 10.1.1.1 Non-neoplastic effects a) Human data i) Skin, connective tissue and bone Among the non-neoplastic effects of vinyl chloride (VC) exposure, disorders of the skin, connective tissue and bone are the most specific and well-characterized. These include acroosteolysis, Raynaud’s phenomenon and sclerodermoid skin lesions. These conditions were quite common in early studies, which may have involved exposures to high levels of several hundred ppm. Acroosteolysis occurred primarily in PVC production workers who had been involved in reactor cleaning. Data on mortality from connective tissue disorders, a relatively rare category of death, have not been provided in published studies, nor are there sufficient data to estimate an exposure–response relationship. ii) Non-neoplastic liver disease Non-neoplastic liver disease was also well-documented in early studies of workers with high levels of exposure to VC. In one carefully described study, “advanced portal hypertension” with histological findings of non-cirrhotic fibrosis was diagnosed in 17 out of 180 VC polymerization workers. Histological alterations reported in liver biopsy specimens obtained from VC monomer workers in another series included focal hepatocytic hyperplasia and focal mixed hyperplasia; these lesions were infrequent in the comparison group. Reversible changes in liver function tests have been reported in VC workers exposed to 2.6–54 mg/m3 (1–21 ppm). Despite evidence for the induction of non-malignant liver disease from clinical studies, there has been no evidence of excess mortality from studies of large cohorts. There are also insufficient data to estimate exposure response. 231 EHC 215: Vinyl Chloride ______________________________________________________ iii) Respiratory disease There is some evidence for respiratory effects from both morbidity and mortality studies of VC workers, but this may be related to PVC- resin dust rather than VC monomer.