Overview
EN ISO 10712:1995 is an international standard published by the European Committee for Standardization (CEN) that specifies a reliable test method for assessing the inhibitory effect of water samples on the growth of Pseudomonas putida. This standard focuses on the Pseudomonas putida growth inhibition test, also known as the Pseudomonas cell multiplication inhibition test, used to evaluate biological water quality.
Its scope covers the testing of surface water, groundwater, and waste water samples to determine potential toxic effects on the bacterium Pseudomonas putida, a gram-negative, aerobic rod-shaped microorganism commonly found in freshwater and soil environments. This test is essential for screening water toxicity and evaluating substances soluble in water. However, it is unsuitable for highly coloured samples or samples containing undissolved or volatile substances that may interfere with nutrient solutions or undergo biochemical degradation during testing.
Key Topics
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Purpose and Application
The standard describes a procedural method for quantifying growth inhibition of Pseudomonas putida after exposure to different concentrations of water samples. Growth is measured by optical density (turbidity), enabling determination of the substance concentrations causing 10% (EC10) and 50% (EC50) inhibition of cell multiplication.
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Test Organism
Utilizes Pseudomonas putida strains (e.g., MIGULA Berlin 33/Z strain DSM 50026 or NCIB strain 9494) as representative heterotrophic freshwater bacteria sensitive to toxicants.
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Test Procedure
- Preparation of stock and precultures using defined nutrient solutions.
- Exposure of test cultures to serial dilutions of the water sample.
- Incubation at 23 ±1 °C in the dark for 16 ±1 hours.
- Measurement of bacterial growth by turbidity using spectrometry or turbidimetry at 436 nm.
- Calculation of percentage inhibition compared to control samples without the test water.
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Sample Requirements and Limitations
Suitable for samples free from interferences such as strong coloration or volatile/undissolved substances. Samples must be thoroughly mixed and tested preferably soon after collection; preservation by cooling or freezing is only recommended under exceptional conditions.
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Reagents and Media
Essential nutrient solutions include sodium nitrate, potassium phosphate salts, yeast extract, glucose, magnesium sulfate, and iron(III) citrate. Stock cultures are maintained on slant agar media as per standard specifications.
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Validity and Quality Control
The method requires parallel replicates for statistical confidence. Inoculum turbidity and culture conditions need precise control to ensure reproducibility. Control and test cultures are compared to assess the growth inhibition.
Applications
The EN ISO 10712:1995 test method is widely used in:
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Environmental Monitoring
Assessing the toxicity of surface, ground, and wastewater to ensure compliance with environmental regulations protecting aquatic life and ecosystem health.
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Water Treatment Facilities
Screening incoming raw water or effluent for toxic substances that could impact microbial treatment processes or downstream environments.
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Chemical and Pharmaceutical Industry
Evaluating the ecotoxicological impact of water-soluble substances and effluents to ensure safe discharge and environmental compatibility.
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Research and Development
Studying the effects of pollutants, disinfectants, or novel compounds on freshwater bacterial populations.
This bacterial growth inhibition test offers a practical, sensitive bioassay for water quality, complementing physicochemical analyses by detecting biologically relevant toxicity effects.
Related Standards
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ISO 7027:1990 – Water quality - Determination of turbidity, which is used within EN ISO 10712 for measuring bacterial cell suspension optical density.
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EN ISO 5667 series – Water quality sampling standards that guide proper collection and handling of water samples to be tested.
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ISO 19438 – Water quality - Determination of inhibition of bacterial growth by other microbial species, offering complementary bioassays in water toxicity testing.
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OECD Guidelines for Testing of Chemicals – Includes bacterial growth inhibition tests using other bacteria such as Vibrio fischeri, often used alongside Pseudomonas tests for comprehensive ecotoxicity assessment.
Keywords
Water quality testing, Pseudomonas putida, bacterial growth inhibition, EC10, EC50, toxicity bioassay, surface water quality, groundwater toxicity test, wastewater monitoring, biological water analysis, ISO 10712, CEN standards, microbiological water quality.
