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| 高通量发光细菌急性毒性试验的实验室间比对 |
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张瑛, 刘亚楠, 陈晓倩
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上海市检测中心
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| 摘要: |
| 我国国家标准GB/T 15441-1995《水质急性毒性的测定发光细菌法》中要求使用剧毒物质氯化汞作为毒性参照物，不仅在实验过程中对实验人员的健康不利，在进入环境后将进一步危害人类健康及生态环境，而且由于该物质管控严格，实验室很难获得，以至于环境监管部门和企业很难以氯化汞作为毒性参照物进行试验，并对水质毒性进行评价，已难以满足管理部门和企业的需要。同时，传统的发光细菌毒性测试方法均是根据孔式化学发光检测仪设计的，每次只能测试1个样品管，将耗费大量的时间和人力，导致通量较低，每次可以测试的样品数量有限，不利于对大量样品的毒性筛查。本研究选择费氏弧菌作为测试菌种，基于96孔板建立了新的高通量、快速、检测灵敏、操作简便、准确评价各类水质毒性的发光细菌毒性测定方法，并对方法的重复性和再现性进行了验证，为该方法的应用研究提供基础数据，为该方法在监测水质毒性应用中的适用性和可行性提供技术支撑，为制定合理的水质标准和废水排放标准提供科学依据。在方法重复性研究中，使用参照物重铬酸钾、七水硫酸锌、3,5-二氯苯酚及三个水样进行测试，重复测定5次，结果显示，参照物重铬酸钾、七水硫酸锌和3,5-二氯苯酚EC50的平均值()分别为33.48、3.53和4.13 mg·L-1，标准偏差(SD)分别为2.79、0.36和0.47 mg·L-1。水样H的EC50均小于样品最低测试浓度(3.13%)，水样M的EC50的和SD分别为22.26%和1.96%，水样L的EC50均大于样品最高测试浓度(80%)。结果表明，重铬酸钾、3,5-二氯苯酚、七水硫酸锌和水样M的5次试验的EC50均在其±2SD范围内。在方法再现性研究中，六家外部实验室使用三种参照物及H、M、L三个水样进行实验室间的比对试验，结果显示，参照物重铬酸钾、七水硫酸锌和3,5-二氯苯酚EC50的分别为31.18、2.80和3.65 mg·L-1，SD分别为8.40、0.51和0.63 mg·L-1。水样H的EC50均小于样品最低测试浓度(3.13%)，水样M的EC50的和SD分别为24.59%和4.06%，水样L的EC50均大于样品最高测试浓度(80%)。结果表明，各实验室重铬酸钾、七水硫酸锌、3,5-二氯苯酚和水样M的EC50均在其±2SD范围内，|z|均<2.0，实验室间的比对结果与总体情况无明显差异，且灵敏度与处于同一营养级的藻类相似，而高于鱼类，低于溞类。因此高通量发光细菌法的水质毒性测试结果具有良好的平行性、重复性及再现性，敏感度较高，适用于生活饮用水、环境水体、污废水等水质的监测，可及时、准确地对水质的急性毒性进行大批量筛查与评估，且可以利用最低无效应稀释度(LID)、效应浓度(EC)和毒性单位(TU)来表征水样的急性毒性水平，并评价毒性大小。 |
| 关键词: 发光细菌 高通量 水质毒性 重复性 再现性 |
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| Interlaboratory Comparison of High-throughput luminescent Bacteria Acute Toxicity Test |
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| Abstract: |
| China's national standard GB/T 15441-1995 "Water quality-Determination of the acute toxicity-Luminescent bacteria test" requires the use of poisonous substance mercury chloride as a toxicity reference, not only adverse to the health of the researchers in the process of experiment, after entering the environment will further harm human health and ecological environment, and due to the strict control of the substance, the laboratory is difficult to obtain. As a result, it is difficult for environmental regulatory departments and enterprises to conduct tests on mercury chloride as a toxicity reference and evaluate the toxicity of water quality, which has been difficult to meet the needs of management departments and enterprises. At the same time, the traditional luminescent bacteria toxicity test methods are based on the single tube chemiluminescence detector design, each time can only test one sample tube, will consume a lot of time and manpower, resulting in low throughput, each can test a limited number of samples, is not conducive to the toxicity screening of a large number of samples. In this study, Aliivibrio fischeri was selected as the test strain and a new luminescent bacteria toxicity determination method with high throughput, rapid detection, sensitive detection, simple operation and accurate evaluation of various water toxicity was established based on 96-well plates, and the repeatability and reproducibility of the method were verified. It provides basic data for the application research of the method, provides technical support for the applicability and feasibility of this method in monitoring the toxicity of water, and provides scientific basis for formulating reasonable water quality standards and wastewater discharge standards. In the method repeatability study, the reference material potassium dichromate, zinc sulfate heptahydrate, 3, 5-dichlorophenol and three water samples were tested and repeated for 5 times. The results showed that the mean values () of EC50 for potassium dichromate, zinc sulfate heptahydrate and 3, 5-dichlorophenol were 33.48, 3.53 and 4.13 mg·L-1, respectively, with standard deviations (SD) of 2.79, 0.36 and 0.47 mg·L-1, respectively. The EC50 of water sample H was lower than the minimum test concentration (3.13%), the and SD of EC50 for water sample M were 22.26% and 1.96%, respectively, and the EC50 of water sample L was higher than the maximum test concentration (80%). The results showed that EC50 of potassium dichromate, 3, 5-dichlorophenol, zinc sulfate heptahydrate and water sample M were all in the range of ±2SD. In the method reproducibility study, six external laboratories conducted inter-laboratory comparison tests using three reference materials and three water samples H, M and L. The results showed that the of EC50 for reference materials potassium dichromate, zinc sulfate heptahydrate and 3, 5-dichlorophenol were 31.18, 2.80 and 3.65 mg·L-1, respectively, with SD of 8.40, 0.51 and 0.63 mg·L-1, respectively. The EC50 of water sample H was lower than the minimum test concentration (3.13%), the and SD of EC50 for water sample M were 24.59% and 4.06%, respectively, and the EC50 of water sample L was higher than the maximum test concentration (80%). The results showed that the EC50 values of potassium bichrodate, zinc sulfate heptahydrate, 3, 5-dichlorophenol and water sample M in all laboratories were in the scope of their ±2SD, and |z| were all <2.0. There was no significant difference between the laboratory comparison results and the general situation, and the sensitivity was similar to that of algae at the same trophic level, but higher than that of fish and lower than that of daphnia. Therefore, the water toxicity test results of high-throughput luminescent bacteria method have good parallelism, repeatability and reproducibility, and are highly sensitive. It is suitable for the monitoring of drinking water, environmental water, waste water and other water quality, and can timely and accurately conduct mass screening and assessment of acute toxicity of water quality. Moreover, the acute toxicity level of water samples can be characterized by lowest ineffective dilution (LID), effect concentration (EC) and toxic unit (TU), and the toxicity can be evaluated. |
| Key words: luminescent bacteria high throughput water quality toxicity repeatability reproducibility |
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