Use of Duckweed (Lemna minor L.) growth inhibition test to evaluate the toxicity of acrylonitrile, sulphocyanic sodium and acetonitrile in China

Environmental Pollution, Vol. 98, No. 2, pp. 143±147, 1997 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain PII: S0269-7491(97)00156-5 0269-7491/97 $17.00+0.00

USE OF DUCKWEED (LEMNA MINOR L.) GROWTH INHIBITION TEST TO EVALUATE THE TOXICITY OF ACRYLONITRILE, SULPHOCYANIC SODIUM AND ACETONITRILE IN CHINA Zhang Tonga and Jin Hongjunb a

Department of Environmental Engineering, East China University of Science and Technology, Shanghai, 20027, China b Department of Environmental Science, Nanjing University, Nanjing, 210008, China (Received 30 September 1996; accepted 26 September 1997)

Abstract This study was designed to evaluate the acute toxicity of three major pollutants (acrylonitrile, sulphocyanic sodium and acetonitrile) in acrylonitrile-acrylic e‚uents to Lemna minor using the growth inhibition test. As well, the study wanted to determine if L. minor can be used as an e€ective bioassay organism in China. The experimental approach involved exposure of L. minor under standard laboratory conditions to the three chemicals. The results indicated that the 96 h±IC50s of the three chemicals to L. minor were 27.08 mg litreÿ1, 3663 mg litreÿ1 and 3685 mg litreÿ1, respectively, while using the relative growth rate as the IC50 endpoint. L. minor proved to be a practical bioassay organism because the L. minor toxicity test is simple, sensitive, and cost-e€ective. # 1998 Elsevier Science Ltd. All rights reserved

useful for stable samples (such as wastewater mainly containing metal ions), but is also suitable for unstable samples (such as wastewater mainly containing volatile organic chemicals) by using ¯ow-through or renewal test modes (Taraldsen and Norberg-King, 1990; Wang, 1991b). Algae toxicity tests cannot be conducted with either of the above modes (Wang, 1991b). Furthermore it is dicult to evaluate the phytotoxicity of wastewater containing background algae with the algae test because the background algae should be removed from the wastewater. These drawbacks are not encountered in duckweed tests using renewal test solution, and the use of non-axenic culture does not alter toxicity test results with L. minor (Taraldsen et al., 1990). Duckweed is ubiquitous in China both in the south and the north, but there are no reports about duckweed bioassay tests in China. Acrylonitrile, acetonitrile and sulphocyanic sodium (NaSCN) are the main pollutants in petroleum chemical industry wastewater, a major kind of industrial wastewater in China. Acrylonitrile has obtained worldwide attention in recent years due to its high toxicity to aquatic life and its carcinogenicity. It is recognized as a priority pollutant in aquatic environments in China as well as in other countries, such as the United States, The Netherlands and Germany (National EPA of China, 1989). Acrylonitrile and acetonitrile, being volatile chemicals, will partition to the air±water interface, which duckweed occupies. However, few reports about the phytotoxicity data of the three pollutants are found in literature. We studied the phytotoxicity of the above three pollutants, using the L. minor renewal test method. The objectives of this study were to determine the phytoxicity of these chemicals and whether duckweed toxicity tests can be e€ectively used as a bioassay method in China. The measurement endpoints used in duckweed toxicity tests include the reduction of frond reproduction and root length, 14C uptake, total Kjeldahl nitrogen and chlorophyll (Fankhauser et al., 1976; Sahai et al., 1977; Filbin and Hough, 1979; Strother, 1980; Huber et al., l982; Rao et al., 1985; Weinberger and Caux, 1985).

Keywords: Lemna minor, phytotoxicity, bioassay, acrylonitrile, acetonitrile. INTRODUCTION Algae have been used as the main test organisms to evaluate the phytotoxicity of pollutants. Plants other than algae, however, should be included in phytotoxicity studies to understand the e€ect of pollutants on multi-cell organisms. Duckweed species, such as Lemna minor, Lemna gibba and Lemna perpusilla, are recommended for aquatic phytotoxicity assessment to complement current phytotoxicity tests using algae (King and Coley, 1985; Caux, 1986; Wang, 1986a,b). Duckweed is a widespread, free¯oating aquatic macrophyte, a source of food for waterfowl and a shelter for small aquatic invertebrates (Scherer, 1979). It grows quickly and reproduces faster than other vascular plants (Wang, 1986a). Although the duckweed toxicity test was proposed in the early 1950s, it became more important in recent years (Taraldsen and Norberg-King, 1990; Wang, 1991a,b). Many reports have shown that the duckweed toxicity test is simple, sensitive, and cost-e€ective (Wang and Williams, 1988, 1990; Wang, 1990a,b). It is not only 143

144

Zhang Tong, Jin Hongjun

Frond number and chlorophyll A were used in this study because they are the indicators of plant biomass and photosynthetic activity, and are simple to measure (Rao et al., 1985; Weinberger and Caux, 1985). MATERIALS AND METHODS Test chemicals Acrylonitrile, sulphocyanic sodium and acetonitrile of analytical grade were purchased from Shanghai Chemical Regent Trade Center, Shanghai, China. L. minor culture L. minor was cultured at 25 C in an aquarium using the culture solution recommended by the US EPA (US EPA, 1989) under continuous illumination of 11 000 Lux light from an overhead ¯uorescent lamp. The culture condition was non-axenic. L. minor fronds of similar size and shape were selected for exposure. Test design The culture solution was used as the dilution water to prepare test solutions. During tests with acetonitrile and NaSCN, 40 ml test solutions were placed in culture dishes with covers (diameter 9 cm). During acrylonitrile tests, 100 ml test solutions were placed in 500 ml Erlenmeyer ¯asks covered with cotton stoppers to reduce the volatilization. There were 4, 4 and 5 test concentrations for acrylonitrile, acetonitrile and NaSCN, respectively (Table 1), and one control group for each test. Every treatment group had four replicate containers. Ten fronds were added to each container and cultured under the same light as that in pre-culturing. The test was performed at 27+2 C for acetonitrile and NaSCN, and at 23+2 C for acrylonitrile because acrylonitrile is Table 1. L. minor growth. The values listed represent the average number of fronds at the end of 4-d renewal tests. RGR is the relative growth rate of L. minor. I is the growth inhibition percent Pollutant

Average SD2 Chemical concentration (mg litreÿ1)

RGR (hÿl)

I (%)

NaSCN

0 696 1080NOEC 1680LOECa 2560a 3920a

29 27 26.5 20.5 18.5 16.75

3.33 14.00 15.00 6.33 3.66 1.58

0.0111 0.0103 0.0102 0.0075 0.0064 0.0054

0 6.3 8.1 32.0 42.0 51.0

Acetonitrile

0 1000NOEC 1800LOECa 3200a 5600a

23 20.25 18 16.5 12.25

12.67 2.25 8.67 3.00 8.91

0.0087 0.0073 0.0061 0.0052 0.0021

0 15 29 40 76

Acrylonitrile

0 6.2NOEC 12.5LOECa 25a 50a

26.75 27.5 22.75 17.25 11

3.75 3.67 6.91 2.25 1.33

a

0.0102 0 0.0105 0 0.0086 16.7 0.0056 44.7 0.00099 90.2

Signi®cantly ( ˆ 0:05) di€erent from the control.

more volati1e than other compounds. Test solutions were renewed daily in acrylonitrile and acetonitrile tests, and every other day in NaSCN tests because NaSCN is more stable than acrylonitrile and acetonitrile. The number of fronds was counted every day for 4 days. In order to eliminate subjective decisions, every frond visibly projecting beyond the edge of the parent frond was counted. As fronds age and die, they lose their pigment and become chlorotic. The chlorotic fronds were not included in the total frond (Hughes et al., 1988; Taraldsen and Norberg-King, 1990). The 4 day test period for duckweed was chosen in this study in order to (1) be comparable to those acute endpoints, (2) avoid overgrowing, and (3) avoid competition by algae. Certainly, 7 days, 14 days or longer period tests might also be performed under careful design in order to study the chronic toxicity of pollutants on duckweed (King and Coley, 1985; Wang, 1985, 1991b). After 96 h exposure, for acetonitrile and NaSCN tests, all the fronds in one container were put into a glass grinder containing 4 ml 90% acetone (10% distilled water). The homogenate was transferred into one centrifuge tube of 10 ml after being grounded for 5 min, then 1 ml 90% acetone was added, and the solution was refrigerated for 72 h at 4 C to extract chlorophyll A. The homogenate was then centrifuged at 2000 rev minÿ1 for 10 min. The optical density (OD) values of the supernatant at 663 nm, 645 nm, 630 nm and 750 nm, respectively, were measured using a quartz glass curette with l cm thickness by a spectrophotometer (model 721, Shanghai Science Instrument Co., Shanghai, China). Data analysis The inhibition percent relative growth rate was selected as the endpoint for biomass response to the toxicants. This endpoint has been used in the algae test and Lemna tests by other researchers (King and Coley, 1985; Wang, 1990b). It was calculated using the following formula (Harper, 1977): Relative Growth rate (RGR)=[Ln(Nt/N0)]/96 h Inhibition percent (I)=(RGR0ÿRGRn)/RGR0100% where: Nt=the average number of fronds per replicate in one treatment group after 96 h exposure N0=the number of fronds per replicate at beginning RGR0=the growth rate of the control group RGRn=the growth rate of the concentration group

Concentration (log10) was plotted against inhibition (%) to determine the 96 h EC50 values. The line of best ®t and the concentration corresponding to 50% inhibition were calculated by inverse linear regression. The chlorophyll A amount in every test container was indicated by the concentration of chlorophyll A in supernatant (Ca). Ca was calculated with the following formula (Zhou, 1990): Ca…mg litreÿ1 † ˆ 11:64  OD663 ÿ 2:16  OD645 ‡ 0:10  OD630 ‡ 9:58  OD750

Use of duckweed (Lemna minor L.) growth inhibition test to evaluate toxicity No observable e€ective concentration (NOEC) is the highest tested concentration where no signi®cant e€ects are observed. Lowest observable e€ective concentration (LOEC) is the lowest concentration, which causes an observed e€ect that is signi®cantly di€erent from control. Samples were independent in this study. The data were ®rst tested for normality, and then tested for homogeneity of variance across all concentrations and controls using Bartlett's method (US EPA, 1988, 1989). Then Dunnett's procedure was performed on homosecastic data to identify the signi®cance (a ˆ 0:05) di€erence, and calculate the NOECs and LOECs.

145

Fig. 2. The relationship of acetonitrile concentration and the inhibition percentages of duckweed relative growth rate (r ˆ 0:955; slope ˆ 77:0; Y-intercept ˆ ÿ221).

RESULTS The data in this study were normally distributed and had homogeneous variance. The relative growth rate inhibition percents of L. minor for the three chemicals are expressed in Figs. 1±3. The 96 h-IC50s of acrylonitrile, acetonitrile and NaSCN were 27.08, 3685 and 3663 mg litreÿ1, respectively. According to phytotoxicity classi®cation suggested by Wang (1986b), acrylonitrile had medium toxicity to L. minor like Ba and Mn ions. The order of toxicity of the three chemicals to L. minor was the same as that to aquatic animals according to the author's test results (Zhang, 1995, 1996a,b,c,d,e, 1997a). The toxicity of all the three pollutants comes from the CNÿ formed from themselves. Acrylonitrile is more toxic because it is easy to produce CNÿ in the solution through hydrolysis than the other two pollutants. Among these growth response curves, the curve for acrylonitrile is the steepest, followed by that of acetonitrile and of NaSCN. These growth response curves are in general agreement with the concentration±response curves of other chemicals (such as atrazine and unionized NH3) for duckweed, including L. minor, L. gibba and L. perpusilla (King and Coley, 1985; Hughes et al., 1988; Wang, 1990b). The curve for acrylonitrile and acetonitrile are both steeper than that for atrazine, which acts as an inhibitor of the photosynthetic process. In groups with high concentrations of pollutants, L. minor colonies became yellow, the roots decayed and the two fronds of the colony separated from each other.

Fig. 3. The relationship of NaSCN concentrations and the inhibition percentages of duckweed relative growth rate (r ˆ 0:971; slope ˆ 58:1; Y-intercept ˆ ÿ158).

DISCUSSION Quality assurance Several quality assurance requirements for duckweed growth inhibition test 1 were suggested by Wang (1990b). This paper proposes the RGRs of duckweed and the deviation of frond numbers among replicates in the control group as two indicators of test quality. Low RGRs indicate incorrect culture condition, whereas large deviations can lead to problems in the reproductivities of the test results. The latter can be Table 2. Concentration of chlorophyll a of L. minor in the supernatant for the NaSCN and acetonitrile tests. The values listed represent the average chlorophyll a concentration per replicate at the end of daily renewal tests Pollutant

NaSCN

Acentonitrileb

a

Fig. 1. The relationship of acrylonitrile concentrations and the inhibition percentages of duckweed relative growth rate (r ˆ 0:977; slope ˆ 98:7; Y-intercept ˆ ÿ85:2).

Chemical Concentration (mg litreÿ1) 0 696 1080 1680 2560NOEC 3920LOECa 0 1000NOEC 1800LOECa 3200a

Average

SD2

1.228 1.179 1.235 1.074 1.127 0.971 0.801 0.670 0.624 0.508

0.021 0.011 0.008 0.033 0.022 0.007 0.012 0.006 0.008 0.003

Signi®cantly (=0.05) di€erent from the control. The chlorophyll A concentration for the acetonitrile concentration above 5600 mg litreÿ1 is not given due to chlorosis of the fronds in such concentrations.

b

146

Zhang Tong, Jin Hongjun

recti®ed by increasing the number of initial fronds for the inoculum. The RGRs of L. minor in control groups were 0.0072±0.0114 hÿ1 in 4-day tests conducted by several researchers (Wang, 1986a; Taraldsen and NorbergKing, 1990). The average RGR of duckweed in control groups of this study was 0.0100 hÿ1. The RGRs in control groups were 0.0102 hÿ1, 0.0087 hÿ1, and 0.0111 hÿ1 for acrylonitrile, acetonitrile and NaSCN, respectively. The RGRs of L. minor in control groups during 96 h tests should be more than 0.0072 hÿ1 according to the results of other researchers (Wang, 1986a; Taraldsen and Norberg-King, 1990) and the authors of this paper. The deviations of frond numbers in control groups in this study were 7.2, 15 and 6% for aclylonitrile, acetonitrile and NaSCN with an initial frond inoculum number of 10. The results of other studies indicated that the deviation of frond numbers in control groups was 18% with an initial frond inoculum number of 20, and was 9% with an initial inoculum of 40 (Wang, 1986a). An inoculum of 10 per replicate would assure sound quality in the L. minor growth inhibition test. Comparison of physiological responses in the L. minor test According to the results of Taraldsen (Taraldsen and Norberg-King, 1990), ChVs determined by chlorophyll A and frond production were same in 88% tests of both e‚uent and single pollutant tests, while chlorophyll A was a more sensitive measurement endpoint than frond production in the remaining tests. In this study the two indicators had the same sensitivity to acetonitrile (Tables 1 and 2). Chlorophyll A was less sensitive to NaSCN than frond production. Thus it is suggested that for practical purposes the chlorophyll analysis be omitted and only frond number be considered in daily tests. Comparison of endpoints Two summary statistics were used in this study. Compared with the EC50, the NOEC is sensitive to the statistical method, the level of signi®cance, the sample size and the selected concentration of test chemicals. Knowledge of 4-day NOEC in this study is useful, but it does not represent a safe concentration at all cases. On the other hand, the EC50 is an appropriate endpoint to use when comparing relative toxicity of various test chemicals or the relative sensitivities of di€erent aquatic plants (Hughes et al., 1988). The 96 h-IC50 of acetonitrile to the growth of the green alga (Scenedesmus quadricauda) was 7300 mg litreÿ1 (Karel, 1983), while the 96 h-IC50 of acetonitrile to L. minor was 368 mg litreÿ1 in this study. The NOEC of NaSCN to S. quadricallda was 120 mg litreÿ1 according to the result of another research performed by the author of this paper, while the NOEC of NaSCN to L. minor was 1080 mg litreÿ1 in this study. This indicated that the relative sensitivity of S. quadricauda and L. minor changed if the pollutants were di€erent.

Comparison between aquatic animals and L. minor Sometimes it is useful to compare EC50 (IC50) values of aquatic plants to EC50 (or LC50) values of aquatic animals. However, it should be remembered that half of the aquatic plant population was inhibited but not killed at the EC50. Taraldsen had compared the sensitivities of L. minor and frequently used test species with single pollutants, industrial wastewaters and e‚uents from wastewater treatment plants (Taraldsen and Norberg-King, 1990). The results indicated that L. minor was as sensitive as other frequently used species for most chemicals and that it was the most sensitive indicator for some industrial wastewaters (Taraldsen and Norberg-King, 1990). Wang studied the toxicity of metal ions to L. minor and concluded that the EPA water quality standards of some metal ions derived from the aquatic animal toxicity tests were not protective for aquatic plants such common duckweed (Wang, 1986a). He proposed to perform phytotoxicity tests to complement aquatic animal tests in developing water quality criteria (Wang, 1986a) and US EPA (1985) also gave out the similar suggestion in their guideline. The authors of this paper studied the toxicity of the three pollutants to aquatic animals, including ®sh, daphnia, larvae of amphibians and larvae of chironomus (Zhang, 1995, 1996a,b,c,d,e, 1997a,b,c,d ). The results (Table 3) indicated that the L. minor test could be used in a battery of standard toxicological tests to increase the knowledge on toxicity pro®le for one chemical. Currently the common bioassay for phytotoxicity of chemicals in water in China is the alga growth inhibition test. The growth response of an algae culture to pollutants is determined either by direct microscopic counts or by indirect spectrophotometer measures. In China, cell counting can be tedious and time-consuming, while the spectrophotometer technique is subject to interference from pollutants or pigment changes in the alga which are not directly related to the growth e€ect. The duckweed test will not only increase our knowledge of the e€ects of pollution on aquatic plants, but will also overcome the shortcomings of algae tests. Our results show that duckweed has the potential to be a good test organism for phytotoxicity in China and can also be used with the traditional algae response methods. Additional tests are necessary to establish a standard duckweed test method for biomonitoring and bioassaying water pollutants in China. Table 3. Comparison between aquatic animals and L. minor (mg litreÿ1) Chemicals

Acrylonitrile Acetonitrile NaSCN

96 h-LC50 of 96 h-IC50 of 48 h-LC50 of aquatic animals L. minor aquatic animals (fish, larvae (daphnia, larvae of amphibian) of chironomus) 5.16±19.6 2400±13 810 104±1691

27.08 3663 3685

10.3±14.2 3160±3562 349

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